Imidazothiazole Derivatives Having Proline Ring Structure

ABSTRACT

Compounds that inhibit interaction between murine double minute 2 (Mdm2) protein and p53 protein and that exhibit anti-tumor activity are provided. Compounds provided by the present disclosure include imidazothiazole derivatives that inhibit interaction between Mdm2 protein and p53 protein and exhibit anti-tumor activity.

TECHNICAL FIELD

The present invention relates to a compound having a proline ringstructure having anti-tumor activity by inhibition of murine doubleminute 2 (Mdm2) or a salt thereof.

BACKGROUND ART

p53 is known as an important factor for inhibiting canceration of cells.p53 is a transcription factor that induces the expression of genesinvolved in the cell cycle and cellular apoptosis in response to variousstresses. p53 is thought to inhibit canceration of cells by atranscription regulating function thereof. In fact, deletion or mutationof the p53 gene is observed in about half of human cancer cases.

Meanwhile, overexpression of murine double minute 2 (Mdm2), a type of E3ubiquitin ligase, is known as a factor for canceration of cells that arecancerated in spite of the presence of normal p53. Mdm2 is a protein ofwhich expression is induced by p53. Mdm2 negatively regulates p53 bymediating degradation of p53 by binding to the transcription activitydomain of p53 to decrease the transcription activity of p53, exportingp53 out of the nucleus, and further acting as a ubiquitination ligaseagainst p53. Therefore, it is thought that inactivation of functions ofand degradation of p53 are promoted in cells in which Mdm2 isoverexpressed, resulting in canceration (Non Patent Document 1).

Paying attention to such functions of Mdm2, many approaches have beenattempted using substances that inhibit the suppression of p53 functionsby Mdm2, as candidate anti-tumor agents. Examples of the Mdm2 inhibitorstargeting the Mdm2-p53 binding site have been reported, which includeimidazoline derivatives having two sites substituted withhalogenobenzene (for example, refer to Non Patent Documents 1 and 2 andPatent Documents 1 to 8) and imidazothiazole derivatives having twosites substituted with halogenobenzene (for example, refer to PatentDocument 9). However, no report has demonstrated that these compoundsactually showed efficacy in clinical practice.

CITATION LIST Patent Documents

-   [Patent Document 1] WO2003/51359-   [Patent Document 2] WO2003/51360-   [Patent Document 3] WO2005/3097-   [Patent Document 4] WO2005/2575-   [Patent Document 5] WO2005/110996-   [Patent Document 6] WO2005/123691-   [Patent Document 7] WO2007/63013-   [Patent Document 8] WO2008/125487-   [Patent Document 9] WO2008/072655

Non Patent Documents

-   [Non Patent Document 1] Science, 2004, 303, 844-848-   [Non Patent Document 2] Proceedings of the National Academy of    Sciences of the United States of America, 2006, 103, 1888-1893

SUMMARY OF INVENTION Problem to be Solved by the Invention

The present invention provides a novel Mdm2 inhibiting compound.Furthermore, the present invention provides an anti-tumor agentcontaining the Mdm2 inhibiting compound.

Means for Solving the Problem

As a result of extensive studies, the present inventors have found thata compound having a structure represented by the following generalformula (1) or a salt thereof had potent Mdm2 inhibiting activity andaccomplished the present invention.

More specifically, the present invention provides:

[1] A compound represented by general formula (1) or a salt thereof:

wherein

Ar₁ represents a phenyl group which may have one or more substituentsselected from a halogen atom, a cyano group, and a C₁-C₆ alkyl group;

Ar₂ represents a phenyl group which may have one or more substituentsselected from a halogen atom, a C₁-C₆ alkyl group, and a cyano group, ora pyridyl group which may have one or more substituents selected from ahalogen atom, a C₁-C₆ alkyl group, and a cyano group;

R¹ represents a C₁-C₆ alkyl group which may have one or moresubstituents selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, a C₁-C₆ alkanoyl group,and a cyano group, a C₁-C₆ alkanoyl group which may have one or moresubstituents selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, and a cyano group, ahydrogen atom, or a hydroxy group;

R² and R³ each independently represent a C₁-C₆ alkyl group which mayhave one or more substituents selected from a halogen atom, a hydroxygroup, a C₁-C₆ alkoxy group, a carbamoyl group, an amino group, a C₁-C₆alkanoyl group, and a cyano group, a carboxy group, or a hydrogen atom,or R² and R³ may together form an oxo group, or R² and R³ together withthe carbon atoms to which R² and R³ are respectively bonded may form a3- to 5-membered saturated hydrocarbon ring in a spiro or condensedform;

R⁴ represents a C₁-C₆ alkyl group which may have one or moresubstituents selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, a C₁-C₆ alkanoyl group,and a cyano group;

R⁵ represents a C₁-C₆ alkyl group which may have one or moresubstituents selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, a C₁-C₆ alkanoyl group,and a cyano group;

R⁶ represents a halogen atom or a hydrogen atom; and

R⁷ represents a halogen atom.

[2] A compound represented by general formula (2) or a salt thereof:

wherein

R¹ represents a C₁-C₆ alkyl group which may have one or moresubstituents selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, a C₁-C₆ alkanoyl group,and a cyano group, a C₁-C₆ alkanoyl group which may have one or moresubstituents selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, and a cyano group, ahydrogen atom, or a hydroxy group;

R² and R³ each independently represent a C₁-C₆ alkyl group which mayhave one or more substituents selected from a halogen atom, a hydroxygroup, a C₁-C₆ alkoxy group, a carbamoyl group, an amino group, a C₁-C₆alkanoyl group, and a cyano group, a carboxy group, or a hydrogen atom,or R² and R³ may together form an oxo group, or R² and R³ together withthe carbon atoms to which R² and R³ are respectively bonded may form a3- to 5-membered saturated hydrocarbon ring in a spiro or condensedform;

R⁴ represents a C₁-C₆ alkyl group which may have one or moresubstituents selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, a C₁-C₆ alkanoyl group,and a cyano group;

R⁵ represents a C₁-C₆ alkyl group which may have one or moresubstituents selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, a C₁-C₆ alkanoyl group,and a cyano group;

R⁶ represents a halogen atom or a hydrogen atom; and

R⁷ represents a halogen atom.

[3] A compound according to [1] or [2] or a salt thereof, wherein R¹ isa C₁-C₆ alkyl group, a C₁-C₆ alkanoyl group which may be substitutedwith one or more halogen atoms, or a hydrogen atom.

[4] A compound according to any one of [1] to [3] or a salt thereof,wherein R⁴ is a C₁-C₆ alkyl group.

[5] A compound according to any one of [1] to [4] or a salt thereof,wherein R⁵ is a C₁-C₆ alkyl group.

[6] A compound represented by the following formula:

[7] A compound represented by the following formula:

[8] A compound represented by the following formula:

[9] A compound represented by the following formula:

[10] A compound represented by the following formula:

[11] An inhibitor of Mdm2 comprising a compound according to any one of[1] to [10] or a salt thereof.

[12] An inhibitor of p53-Mdm2 binding comprising a compound according toany one of [1] to [10] or a salt thereof.

[13] A medicament comprising a compound according to any one of [1] to[10] or a salt thereof as an active ingredient.

[14] A pharmaceutical composition comprising a compound according to anyone of [1] to [10] or a salt thereof and a pharmaceutically acceptablecarrier.

[15] Use of a compound according to any one of [1] to [10] or a saltthereof for the manufacture of a medicament.

[16] An anticancer agent comprising a compound according to any one of[1] to [10] or a salt thereof as an active ingredient.

[17] A method for treating cancer, comprising administering a compoundaccording to any one of [1] to [10] or a salt thereof.

[18] An anticancer agent according to [16], wherein the cancer is anyselected from lung cancer, breast cancer, prostate cancer, colon cancer,acute myeloid leukemia, malignant lymphoma, retinoblastoma,neuroblastoma, and sarcoma.

[19] A method for treating cancer according to [17], wherein the canceris any selected from lung cancer, breast cancer, prostate cancer, coloncancer, acute myeloid leukemia, malignant lymphoma, retinoblastoma,neuroblastoma, and sarcoma.

Advantages of the Invention

The present invention provides a novel imidazothiazole derivativerepresented by the above formula (1), which has Mdm2 inhibitingactivity. Such a novel compound is useful as an anti-tumor agent.

DESCRIPTION OF EMBODIMENTS

In the present invention, “Mdm2” means a protein encoded by the murinedouble minute 2 gene. “Mdm2” includes Mdm2 proteins encoded by acomplete length of the Mdm2 gene, Mdm2 proteins encoded by mutated Mdm2genes (including deletion mutants, substitution mutants, and additionmutants), and so forth. In the present invention, “Mdm2” also includeshomologues derived from various animal species such as, for example,human Mdm2 homologue (HDM2).

In the present invention, “p53” means a protein encoded by the p53 gene.“p53” means the p53 protein encoded by a full length p53 gene or a p53protein that has a mutation (including mutations by deletion,substitution, and addition), but functions normally.

In the present invention, “Mdm2 inhibitor” means a factor that restoresp53 functions suppressed by Mdm2 by acting on either Mdm2 or p53, or onboth p53 and Mdm2. The p53 functions are not particularly limited solong as they are functions which p53 normally has. Examples thereofinclude inhibition of canceration of cells by inducing the expression ofgenes involved in the cell cycle or cellular apoptosis. Examples of Mdm2inhibitors include factors that inhibit binding of Mdm2 to p53(hereinafter, referred to as p53-Mdm2 binding inhibitors) or factorsthat inhibit ubiquitination of p53 by Mdm2 (hereinafter, referred to asMdm2 ubiquitin ligase inhibitors).

In the present invention, “inhibitor of suppression of p53 transcriptionactivity” means a factor that restores the functions of p53 as atranscription factor suppressed by Mdm2.

In the present invention, “inhibitor of p53 degradation” means a factorthat inhibits degradation of p53 in proteasomes by inhibitingubiquitination of p53 by Mdm2.

In the present invention, the terms “tumor” and “cancer” are usedinterchangeably. Furthermore, in the present invention, tumor, malignanttumor, cancer, malignant neoplasm, carcinoma, sarcoma, and the like maybe collectively referred to as “tumor” or “cancer.”

In the present invention, “C₁-C₆ alkyl group” means a straight,branched, or cyclic alkyl group having 1 to 6 carbon atoms. Examples ofa “C₁-C₆ alkyl group” include a methyl group, an ethyl group, a propylgroup, an isopropyl group, a cyclopropyl group, a butyl group, atert-butyl group, a cyclobutyl group, a cyclopentyl group, and acyclohexyl group.

“C₁-C₆ alkoxy group” means an alkoxy group having a straight, branched,or cyclic alkyl group having 1 to 6 carbon atoms. Examples of a “C₁-C₆alkoxy group” include a methoxy group, an ethoxy group, a propoxy group,an isopropoxy group, a butoxy group, and a cyclopentyloxy group.

“C₁-C₆ alkanoyl group” means an alkanoyl group having a straight,branched, or cyclic alkyl group having 1 to 6 carbon atoms. Examples ofa “C₁-C₆ alkanoyl group” include a formyl group, an acetyl group, apropionyl group, and a methylpropionyl group.

Examples of “halogen atom” include a fluorine atom, a chlorine atom, abromine atom, and an iodine atom.

“Oxo group” means a group represented by “═O” unless otherwisespecified.

Hereafter, each substituent in formula (1) will be explained.

In the following general formula (1),

R¹ represents a C₁-C₆ alkyl group which may have one or moresubstituents selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, a C₁-C₆ alkanoyl group,and a cyano group, a C₁-C₆ alkanoyl group which may have one or moresubstituents selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, and a cyano group, ahydrogen atom, or a hydroxy group.

Here, the “C₁-C₆ alkyl group which may have one or more substituents”and the “C₁-C₆ alkanoyl group which may have one or more substituents”preferably have 0 to 3 substituents. The substituent(s) is preferably ahalogen atom, a hydroxy group, a C₁-C₆ alkoxy group, a carbamoyl group,an amino group, a C₁-C₆ alkanoyl group, or a cyano group. Thesubstituent(s) is more preferably a halogen atom, a hydroxy group, or aC₁-C₆ alkoxy group.

The “C₁-C₆ alkyl group which may have one or more substituents” is morepreferably an unsubstituted C₁-C₆ alkyl group or a C₁-C₆ alkyl grouphaving one or more halogen atoms, hydroxy groups, or C₁-C₆ alkoxy groupsas a substituent, yet more preferably an unsubstituted C₁-C₆ alkyl groupor a C₁-C₆ alkyl group having 1 to 3 fluorine atoms as a substituent.

The “C₁-C₆ alkanoyl group which may have one or more substituents” ispreferably an unsubstituted C₁-C₆ alkanoyl group or a C₁-C₆ alkanoylgroup having 1 to 3 halogen atoms as a substituent, particularlypreferably a formyl group, an acetyl group, or a trifluoromethylcarbonylgroup.

R¹ is preferably an unsubstituted C₁-C₆ alkyl group or a C₁-C₆ alkylgroup substituted with halogen atom(s), an unsubstituted C₁-C₆ alkanoylgroup or a C₁-C₆ alkanoyl group substituted with halogen atom(s), or ahydrogen atom.

R² and R³ each independently represent a C₁-C₆ alkyl group which mayhave one or more substituents selected from a halogen atom, a hydroxygroup, a C₁-C₆ alkoxy group, a carbamoyl group, an amino group, a C₁-C₆alkanoyl group, and a cyano group, a carboxy group, or a hydrogen atom,or R² and R³ may together form an oxo group, or R² and R³ together withthe carbon atoms to which R² and R³ are respectively bonded may form a3- to 5-membered saturated hydrocarbon ring in a spiro or condensedform.

Here, the “C₁-C₆ alkyl group which may have one or more substituents”has the same meaning as defined above in R¹ and also has the samepreferred examples.

When both R² and R³ are respectively a carboxy group or one of R² and R³is a carboxy group, compounds in which the carboxy group is esterifiedor amidated are also included in the scope of the present invention.Examples thereof include compounds in which the carboxy group is ethylesterified, phenyl esterified, carboxymethyl esterified,dimethylaminomethyl esterified, pivaloyloxymethyl esterified,ethoxycarbonyloxyethyl esterified, amidated, or methylamidated.

The phrase “R² and R³ together with the carbon atoms to which R² and R³are respectively bonded may form a 3- to 5-membered saturatedhydrocarbon ring in a spiro or condensed form” means that R² and R³ maybe bonded to form a 3- to 5-membered saturated hydrocarbon ring in aspiro form when R² and R³ are substituents bonded to the same carbonatom, or R² and R³ may be bonded to form a 3- to 5-membered saturatedhydrocarbon ring in a condensed form when R² and R³ are bonded todifferent carbon atoms. Examples of the “3- to 5-membered saturatedhydrocarbon ring” include a cyclopropane ring, a cyclobutane ring, and acyclopentane ring.

Preferably, R² and R³ are each independently a hydrogen atom, a C₁-C₆alkyl group which may have substituent(s), or a carboxy group. Thesubstituent substituted on the C₁-C₆ alkyl group is preferably afluorine atom or a hydroxy group. More preferably, R² and R³ arerespectively a hydrogen atom, or one of R² and R³ is a hydrogen atom andthe other moiety is a methyl group or an ethyl group, or R² and R³ whichare C₁-C₆ alkyl groups bonded to the same carbon atom form a 3- to4-membered saturated hydrocarbon ring in a spiro form, together with thecarbon atom to which they are bonded. Both R² and R³ are preferablysubstituents at the 6-position on a piperazine ring.

R⁴ represents a C₁-C₆ alkyl group which may have one or moresubstituents. Here, the “C₁-C₆ alkyl group which may have one or moresubstituents” has the same meaning as defined above in R¹.

R⁴ is preferably an unsubstituted C₁-C₆ alkyl group, more preferably anunsubstituted C₁-C₃ alkyl group, yet more preferably an isopropyl group.

R⁵ represents a C₁-C₆ alkyl group which may have one or moresubstituents. Here, the “C₁-C₆ alkyl group which may have one or moresubstituents” has the same meaning as defined above in R¹.

R⁵ is preferably an unsubstituted C₁-C₆ alkyl group, more preferably anunsubstituted C₁-C₃ alkyl group, yet more preferably a methyl group oran ethyl group.

R⁶ represents a halogen atom or a hydrogen atom. R⁶ is preferably afluorine atom, a chlorine atom, a bromine atom, or a hydrogen atom, morepreferably a fluorine atom or a hydrogen atom.

R⁷ represents a halogen atom. R⁷ is preferably a fluorine atom, achlorine atom, or a bromine atom, more preferably a fluorine atom.

Ar₁ represents a phenyl group which may have one or more substituents.Ar₁ is preferably a phenyl group having 1 to 3 C₁-C₆ alkyl groups, cyanogroups, or halogen atoms as a substituent, more preferably a phenylgroup having 1 to 3 halogen atoms as a substituent. The substituent maybe positioned at any position. More preferably, Ar₁ is a 4-chlorophenylgroup or a 3-fluoro-4-chlorophenyl group.

Ar₂ represents a phenyl group which may have one or more substituents ora pyridyl group which may have one or more substituents. Ar₂ ispreferably a phenyl group having 1 to 3 halogen atoms, C₁-C₆ alkylgroups, or cyano groups as a substituent or a pyridyl group having 1 to3 halogen atoms, C₁-C₆ alkyl groups, or cyano groups as a substituent.The substituent may be positioned at any position. More preferably, Ar₂is a 4-chlorophenyl group or a 6-chloropyridin-3-yl group.

The absolute configurations of Ar₁ and Ar₂ in the imidazothiazoleskeleton are preferably 5R and 6S, respectively.

Furthermore, the compound represented by the formula (1) of the presentinvention is preferably a compound represented by the following generalformula (2):

Here, definitions, examples, and preferred examples of R¹, R², R³, R⁴,R⁵, R⁶, and R⁷ are the same as described above in general formula (1).

Furthermore, the compound of the present invention is preferably onecompound selected from the following group:

The compound represented by formula (1) of the present invention mayhave stereoisomers or optical isomers due to asymmetric carbon atoms,and all these stereoisomers, optical isomers, and mixtures thereof areincluded in the present invention.

In one embodiment of the present invention, a compound having anabsolute configuration represented by formula (3) is preferred:

wherein Ar₁, Ar₂, R¹ to R⁷ have the same meanings as defined above.

Furthermore, the compound of general formula (1) is preferably acompound, a salt or a hydrate thereof described in any of the Examplesand/or Tables 1 to 12 described later.

The compound represented by general formula (1) of the present inventioncan form a pharmaceutically acceptable salt, if desired, when having abasic group such as an amino group. Examples of such salts can include:hydrohalides such as hydrochloride and hydroiodide; inorganic acid saltssuch as nitrate, perchlorate, sulfate, and phosphate; loweralkanesulfonates such as methanesulfonate, trifluoromethanesulfonate,and ethanesulfonate; arylsulfonates such as benzenesulfonate andp-toluenesulfonate; organic acid salts such as formic acid, acetic acid,malic acid, fumarate, succinate, citrate, tartrate, oxalate, andmaleate; and amino acid salts such as ornithine salt, glutamate, andaspartate. Hydrohalides and organic acid salts are preferred.

The compound represented by general formula (1) of the present inventionmay generally form a base addition salt when having an acidic group suchas a carboxy group. Examples of pharmaceutically acceptable salts caninclude: alkali metal salts such as sodium salt, potassium salt, andlithium salt; alkaline earth metal salts such as calcium salt andmagnesium salt; inorganic salts such as ammonium salt; and organic aminesalts such as dibenzylamine salt, morpholine salt, phenylglycine alkylester salt, ethylenediamine salt, N-methylglucamine salt, diethylaminesalt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt,N,N′-dibenzylethylenediamine salt, diethanolamine salt,N-benzyl-N-(2-phenylethoxy)amine salt, piperazine salt,tetramethylammonium salt, and tris(hydroxymethyl)aminomethane salt.

The compound represented by general formula (1) of the present inventionor the salt thereof may be present in a free or solvate form. Thecompound represented by general formula (1) of the present invention orthe salt thereof may be present in a hydrate form, for example, byabsorbing moisture in the air. The solvate is not particularly limitedso long as it is pharmaceutically acceptable. Specifically, the solvateis preferably a hydrate, an ethanol solvate, or the like. Moreover, thecompound represented by general formula (1) of the present invention maybe in an N-oxide form when containing a nitrogen atom. These solvate andN-oxide forms are also included in the present invention.

The compound represented by general formula (1) of the present inventionmay have various isomers such as geometrical isomers (e.g., cis andtrans forms), tautomers, and optical isomers (e.g., d and l forms),depending on the types or combinations of substituents. The compound ofthe present invention also encompasses all these isomers, stereoisomers,and mixtures of these isomers and stereoisomers in any ratio, unlessotherwise specified.

The compound represented by general formula (1) of the present inventionmay contain an isotope in a non-natural proportion as one or moreconstituent atoms. Examples of an isotope include deuterium (²H),tritium (³H), iodine-125 (¹²⁵I), and carbon-14 (¹⁴C). These compoundsare useful as a therapeutic or preventive agent, a research reagent(e.g., an assay reagent), and a diagnostic agent (e.g., an in vivodiagnostic imaging agent). All isotopic variants of the compoundrepresented by general formula (1) are included in the scope of thepresent invention, regardless of the presence or absence ofradioactivity.

Moreover, the present invention also encompasses a compound that isconverted to the compound (1) as an active ingredient in thepharmaceutical composition of the present invention due to a reactioninduced by an enzyme, gastric acid, or the like under physiologicalconditions in vivo, i.e., a compound that is converted to the compound(1) through enzymatic oxidation, reduction, hydrolysis, or the like or a“pharmaceutically acceptable prodrug compound” that is converted to thecompound (1) through hydrolysis or the like induced by gastric acid orthe like.

Examples of a prodrug can include: compounds in which an amino group inthe compound (1) is acylated, alkylated, or phosphorylated (e.g.,compounds in which the amino group is eicosanoylated, alanylated,pentylaminocarbonylated,(5-methyl-2-oxo-1,3-dioxolen-4-yl)methoxycarbonylated,tetrahydrofuranylated, pyrrolidylmethylated, pivaloyloxymethylated, ortert-butylated); compounds in which a hydroxy group in the compound (1)is acylated, alkylated, phosphorylated, or borated (e.g., compounds inwhich the hydroxy group is acetylated, palmitoylated, propanoylated,pivaloylated, succinylated, fumarylated, alanylated, ordimethylaminomethylcarbonylated); and compounds in which a carboxy groupin the compound (1) is esterified or amidated (e.g., compounds in whichthe carboxy group is ethyl esterified, phenyl esterified, carboxymethylesterified, dimethylaminomethyl esterified, pivaloyloxymethylesterified, ethoxycarbonyloxyethyl esterified, amidated, ormethylamidated).

A prodrug of the compound of the present invention can be produced fromthe compound (1) according to a method known in the art. Moreover, aprodrug of the compound of the present invention also includes thoseconverted to the compound (1) under physiological conditions asdescribed in “Development of Pharmaceutical Products”, vol. 7, MoleculeDesign, p. 163-198, Hirokawa-Shoten Ltd. (1990).

Specific examples of the compound represented by general formula (1) ofthe present invention can include compounds described in, for example,the following Compound Tables 1 to 12. These compounds can besynthesized according to [Production Method 1] to [Production Method 4]described later or methods described in the Examples. In the tables, R¹,R², R³, R⁴, R⁵, R⁶, R⁷, Ar₁ and Ar₂ means groups represented by thefollowing general formula (1a).

TABLE 1 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂  1 H H H CH(CH₃)₂ CH₃ H F

 2 H CH₃ H CH(CH₃)₂ CH₃ H F

 3 H CH₃ CH₃ CH(CH₃)₂ CH₃ H F

 4 H CH₂CH₃ H CH(CH₃)₂ CH₃ H F

 5 H CH(CH₃)₂ H CH(CH₃)₂ CH₃ H F

 6 H CH₂CH(CH₃)₂ H CH(CH₃)₂ CH₃ H F

 7 H CH₂OH H CH(CH₃)₂ CH₃ H F

 8 H CO₂H H CH(CH₃)₂ CH₃ H F

 9 H CH₂F H CH(CH₃)₂ CH₃ H F

10 H CHF₂ H CH(CH₃)₂ CH₃ H F

11 H cyclopropyl CH(CH₃)₂ CH₃ H F

12 H cyclobutyl CH(CH₃)₂ CH₃ H F

13 CH₃ H H CH(CH₃)₂ CH₃ H F

14 CH₃ CH₃ H CH(CH₃)₂ CH₃ H F

15 CH₃ CH₃ CH₃ CH(CH₃)₂ CH₃ H F

TABLE 2 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂ 16 CH₃ CH₂CH₃ H CH(CH₃)₂ CH₃ HF

17 CH₃ CH(CH₃)₂ H CH(CH₃)₂ CH₃ H F

18 CH₃ CH₂CH(CH₃)₂ H CH(CH₃)₂ CH₃ H F

19 CH₃ CH₂F H CH(CH₃)₂ CH₃ H F

20 CH₃ CHF₂ H CH(CH₃)₂ CH₃ H F

21 CH₃ cyclopropyl CH(CH₃)₂ CH₃ H F

22 CH₃ cyclobutyl CH(CH₃)₂ CH₃ H F

23 CH₂CH₂OH CH₃ H CH(CH₃)₂ CH₃ H F

24 CHO CH₃ H CH(CH₃)₂ CH₃ H F

25 CHO CH₂CH₃ H CH(CH₃)₂ CH₃ H F

26 COCH₃ CH₃ H CH(CH₃)₂ CH₃ H F

27 COCH₃ CH₂CH₃ H CH(CH₃)₂ CH₃ H F

28 COCF₃ CH₃ H CH(CH₃)₂ CH₃ H F

29 COCF₃ CH₂CH₃ H CH(CH₃)₂ CH₃ H F

30 H H H CH(CH₃)₂ CH₃ F F

TABLE 3 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂ 31 H CH₃ H CH(CH₃)₂ CH₃ F F

32 H CH₃ CH₃ CH(CH₃)₂ CH₃ F F

33 H CH₂CH₃ H CH(CH₃)₂ CH₃ F F

34 H CH(CH₃)₂ H CH(CH₃)₂ CH₃ F F

35 H CH₂CH(CH₃)₂ H CH(CH₃)₂ CH₃ F F

36 H CH₂OH H CH(CH₃)₂ CH₃ F F

37 H CO₂H H CH(CH₃)₂ CH₃ F F

38 H CH₂F H CH(CH₃)₂ CH₃ F F

39 H CHF₂ H CH(CH₃)₂ CH₃ F F

40 H cyclopropyl CH(CH₃)₂ CH₃ F F

41 H cyclobutyl CH(CH₃)₂ CH₃ F F

42 CH₃ H H CH(CH₃)₂ CH₃ F F

43 CH₃ CH₃ H CH(CH₃)₂ CH₃ F F

44 CH₃ CH₃ CH₃ CH(CH₃)₂ CH₃ F F

45 CH₃ CH₂CH₃ H CH(CH₃)₂ CH₃ F F

TABLE 4 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂ 46 CH₃ CH(CH₃)₂ H CH(CH₃)₂ CH₃F F

47 CH₃ CH₂CH(CH₃)₂ H CH(CH₃)₂ CH₃ F F

48 CH₃ CH₂F H CH(CH₃)₂ CH₃ F F

49 CH₃ CHF₂ H CH(CH₃)₂ CH₃ F F

50 CH₃ cyclopropyl CH(CH₃)₂ CH₃ F F

51 CH₃ cyclobutyl CH(CH₃)₂ CH₃ F F

52 CH₂CH₂OH CH₃ H CH(CH₃)₂ CH₃ F F

53 CHO CH₃ H CH(CH₃)₂ CH₃ F F

54 CHO CH₂CH₃ H CH(CH₃)₂ CH₃ F F

55 COCH₃ CH₃ H CH(CH₃)₂ CH₃ F F

56 COCH₃ CH₂CH₃ H CH(CH₃)₂ CH₃ F F

57 COCF₃ CH₃ H CH(CH₃)₂ CH₃ F F

58 COCF₃ CH₂CH₃ H CH(CH₃)₂ CH₃ F F

59 H H H CH(CH₃)₂ CH₃ H F

60 H CH₃ H CH(CH₃)₂ CH₃ H F

TABLE 5 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂ 61 H CH₃ CH₃ CH(CH₃)₂ CH₃ H F

62 H CH₂CH₃ H CH(CH₃)₂ CH₃ H F

63 H CH(CH₃)₂ H CH(CH₃)₂ CH₃ H F

64 H CH₂CH(CH₃)₂ H CH(CH₃)₂ CH₃ H F

65 H CH₂OH H CH(CH₃)₂ CH₃ H F

66 H CO₂H H CH(CH₃)₂ CH₃ H F

67 H CH₂F H CH(CH₃)₂ CH₃ H F

68 H CHF₂ H CH(CH₃)₂ CH₃ H F

69 H cyclopropyl CH(CH₃)₂ CH₃ H F

70 H cyclobutyl CH(CH₃)₂ CH₃ H F

71 CH₃ H H CH(CH₃)₂ CH₃ H F

72 CH₃ CH₃ H CH(CH₃)₂ CH₃ H F

73 CH₃ CH₃ CH₃ CH(CH₃)₂ CH₃ H F

74 CH₃ CH₂CH₃ H CH(CH₃)₂ CH₃ H F

75 CH₃ CH(CH₃)₂ H CH(CH₃)₂ CH₃ H F

TABLE 6 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂ 76 CH₃ CH₂CH(CH₃)₂ H CH(CH₃)₂CH₃ H F

77 CH₃ CH₂F H CH(CH₃)₂ CH₃ H F

78 CH₃ CHF₂ H CH(CH₃)₂ CH₃ H F

79 CH₃ cyclopropyl CH(CH₃)₂ CH₃ H F

80 CH₃ cyclobutyl CH(CH₃)₂ CH₃ H F

81 CH₂CH₂OH CH₃ H CH(CH₃)₂ CH₃ H F

82 CHO CH₃ H CH(CH₃)₂ CH₃ H F

83 CHO CH₂CH₃ H CH(CH₃)₂ CH₃ H F

84 COCH₃ CH₃ H CH(CH₃)₂ CH₃ H F

85 COCH₃ CH₂CH₃ H CH(CH₃)₂ CH₃ H F

86 COCF₃ CH₃ H CH(CH₃)₂ CH₃ H F

87 COCF₃ CH₂CH₃ H CH(CH₃)₂ CH₃ H F

88 H H H CH(CH₃)₂ CH₃ F F

89 H CH₃ H CH(CH₃)₂ CH₃ F F

90 H CH₃ CH₃ CH(CH₃)₂ CH₃ F F

TABLE 7 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂  91 H CH₂CH₃ H CH(CH₃)₂ CH₃ F F

 92 H CH(CH₃)₂ H CH(CH₃)₂ CH₃ F F

 93 H CH₂ CH(CH₃)₂ H CH(CH₃)₂ CH₃ F F

 94 H CH₂OH H CH(CH₃)₂ CH₃ F F

 95 H CH₂H H CH(CH₃)₂ CH₃ F F

 96 H CH₂F H CH(CH₃)₂ CH₃ F F

 97 H CHF₂ H CH(CH₃)₂ CH₃ F F

 98 H cyclopropyl CH(CH₃)₂ CH₃ F F

 99 H cyclobutyl CH(CH₃)₂ CH₃ F F

100 CH₃ H H CH(CH₃)₂ CH₃ F F

101 CH₃ CH₃ H CH(CH₃)₂ CH₃ F F

102 CH₃ CH₃ CH₃ CH(CH₃)₂ CH₃ F F

103 CH₃ CH₂CH₃ H CH(CH₃)₂ CH₃ F F

104 CH₃ CH(CH₃)₂ H CH(CH₃)₂ CH₃ F F

105 CH₃ CH₂CH(CH₃)₂ H CH(CH₃)₂ CH₃ F F

TABLE 8 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂ 106 CH₃ CH₂F H CH(CH₃)₂ CH₃ F F

107 CH₃ CHF₂ H CH(CH₃)₂ CH₃ F F

108 CH₃ cyclopropyl CH(CH₃)₂ CH₃ F F

109 CH₃ cyclobutyl CH(CH₃)₂ CH₃ F F

110 CH₂CH₂OH CH₃ H CH(CH₃)₂ CH₃ F F

111 CHO CH₃ H CH(CH₃)₂ CH₃ F F

112 CHO CH₂CH₃ H CH(CH₃)₂ CH₃ F F

113 COCH₃ CH₃ H CH(CH₃)₂ CH₃ F F

114 COCH₃ CH₂CH₃ H CH(CH₃)₂ CH₃ F F

115 COCF₃ CH₃ H CH(CH₃)₂ CH₃ F F

116 COCF₃ CH₂CH₃ H CH(CH₃)₂ CH₃ F F

117 H CH₃ H CH(CH₃)₂ CH₃ H F

118 H CH₂CH₃ H CH(CH₃)₂ CH₃ H F

119 H CH(CH₃)₂ H CH(CH₃)₂ CH₃ H F

120 H CH₂F H CH(CH₃)₂ CH₃ H F

TABLE 9 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂ 121 H CHF₂ H CH(CH₃)₂ CH₃ H F

122 H cyclopropyl CH(CH₃)₂ CH₃ H F

123 CH₃ CH₃ H CH(CH₃)₂ CH₃ H F

124 CH₃ CH₂CH₃ H CH(CH₃)₂ CH₃ H F

125 CH₃ CH(CH₃)₂ H CH(CH₃)₂ CH₃ H F

126 CH₃ CH₂F H CH(CH₃)₂ CH₃ H F

127 CH₃ CHF₂ H CH(CH₃)₂ CH₃ H F

128 CH₃ cyclopropyl CH(CH₃)₂ CH₃ H F

129 H CH₃ H CH(CH₃)₂ CH₃ F F

130 H CH₂CH₃ H CH(CH₃)₂ CH₃ F F

131 H CH(CH₃)₂ H CH(CH₃)₂ CH₃ F F

132 H CH₂F H CH(CH₃)₂ CH₃ F F

133 H CHF₂ H CH(CH₃)₂ CH₃ F F

134 H cyclopropyl CH(CH₃)₂ CH₃ F F

135 CH₃ CH₃ H CH(CH₃)₂ CH₃ F F

TABLE 10 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂ 136 CH₃ CH₂CH₃ H CH(CH₃)₂ CH₃F F

137 CH₃ CH(CH₃)₂ H CH(CH₃)₂ CH₃ F F

138 CH₃ CH₂F H CH(CH₃)₂ CH₃ F F

139 CH₃ CHF₂ H CH(CH₃)₂ CH₃ F F

140 CH₃ cyclopropyl CH(CH₃)₂ CH₃ F F

141 H CH₃ H CH(CH₃)₂ CH₃ H F

142 H CH₂CH₃ H CH(CH₃)₂ CH₃ H F

143 H CH₃ H CH(CH₃)₂ CH₃ F F

144 H CH₂CH₃ H CH(CH₃)₂ CH₃ F F

145 H CH₃ H CH(CH₃)₂ CH₃ H F

146 H CH₂CH₃ H CH(CH₃)₂ CH₃ H F

147 H CH₃ H CH(CH₃)₂ CH₃ F F

148 H CH₂CH₃ H CH(CH₃)₂ CH₃ F F

149 H CH₃ H CH(CH₃)₂ CH₃ H F

150 H CH₂CH₃ H CH(CH₃)₂ CH₃ H F

TABLE 11 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂ 151 H CH₃ H CH(CH₃)₂ CH₃ F F

152 H CH₂CH₃ H CH(CH₃)₂ CH₃ F F

153 H CH₃ H CH(CH₃)₂ CH₃ H F

154 H CH₂CH₃ H CH(CH₃)₂ CH₃ H F

155 H CH₃ H CH(CH₃)₂ CH₃ F F

156 H CH₂CH₃ H CH(CH₃)₂ CH₃ F F

157 H CH₃ H CH(CH₃)₂ CH₃ H F

158 H CH₂CH₃ H CH(CH₃)₂ CH₃ H F

159 H CH₃ H CH(CH₃)₂ CH₃ F F

160 H CH₂CH₃ H CH(CH₃)₂ CH₃ F F

TABLE 12 No. R¹ R² R³ R⁴ R⁵ R⁶ R⁷ Ar₁ Ar₂ 161 H H H CH(CH₃)₂ CH₃ H F

162 H CH₃ H CH(CH₃)₂ CH₃ H F

163 H CH₂CH₃ H CH(CH₃)₂ CH₃ H F

164 H H H CH(CH₃)₂ CH₃ H F

165 H CH₃ H CH(CH₃)₂ CH₃ H F

166 H CH₂CH₃ H CH(CH₃)₂ CH₃ H F

Next, a representative method for producing a compound represented bygeneral formula (1) will be explained. The compound of the presentinvention can be produced by various production methods and thefollowing production methods are illustrative and should not beconstrued in any limitative way. Reactions shown below can be performedby protecting substituents with appropriate protective groups, ifnecessary, and the types of protective groups are not particularlylimited.

[Production Method 1]

wherein Z means a halogen atom such as a chlorine atom or a bromineatom; R¹¹ means a protective group for the carboxy group; R¹² means aprotective group for the amino group. It is to be noted that these“protective groups” may be the same or different while the reactionsteps are gone through, as long as they are “protective groups”. Ar₁,Ar₂, and R¹ to R⁷ have the same meanings as defined above.

Examples of the protective group for the carboxy group includesubstituted or unsubstituted alkyl groups or aralkyl groups such as amethyl group, an ethyl group, a tert-butyl group, and a benzyl group.

Synthesis of Compound (4)

A compound (4) can be obtained by reacting an optically active diaminecompound (3) having the positional configuration shown above with carbondisulfide or 1,1′-thiocarbonyldiimidazole. Here, the solvent used in thereaction is not particularly limited and examples thereof includeethanol, tetrahydrofuran, dioxane, acetonitrile, dimethylformamide,dichloromethane, chloroform, toluene, and mixed solvents thereof. Thereaction temperature is usually in the range from −78 to 100° C. or theboiling point of the solvent, preferably in the range from around roomtemperature to 100° C.

Synthesis of Compound (5)

A compound (5) can be obtained by reacting compound (4) with a compound(A). Here, the solvent used in the reaction is not particularly limitedand examples thereof include ethanol, tetrahydrofuran, dioxane,acetonitrile, dimethylformamide, chloroform, toluene, and mixed solventsthereof. The reaction temperature is usually in the range from −78 to100° C. or the boiling point of the solvent, preferably in the rangefrom around room temperature to 100° C.

Synthesis of Compound (6)

This step is performed for the deprotection of R¹¹.

Although deprotection reaction conditions differ depending on the typeof R¹¹, R¹¹ may be deprotected by hydrolysis. When R¹¹ is a methylgroup, an ethyl group, a benzyl group, or the like, a compound (6) canbe obtained by treating compound (5) with a base (e.g., sodiumhydroxide, potassium hydroxide, lithium hydroxide, or potassiumtert-butoxide) or hydrochloric acid, p-toluenesulfonic acid, or thelike. Here, examples of the solvent used in the reaction includemethanol, ethanol, water, tetrahydrofuran, dioxane, and mixed solventsthereof. However, organic solvents that can be mixed with water in anarbitrary ratio are preferred. The reaction temperature is usually inthe range from −78 to 100° C. or the boiling point of the solvent,preferably in the range from around room temperature to 100° C. When R¹¹is a tert-butyl group or the like, compound (5) is preferably treatedwith trifluoroacetic acid or hydrochloric acid or the like. Here, thesolvent used in the reaction is not particularly limited and examplesthereof include dichloromethane, chloroform, and mixed solvents thereof.The reaction temperature is usually in the range from −78 to 100° C. orthe boiling point of the solvent, preferably in the range from −20° C.to around room temperature.

Synthesis of Compound (7)

A compound (7) can be obtained by reacting compound (6) with an acidhalogenating reagent such as thionyl chloride, oxalyl chloride,phosphorus oxychloride, or 1-chloro-N,N,2-trimethyl-1-propenylamine.Here, the solvent used in the reaction is not particularly limited andexamples thereof include dichloromethane, chloroform, toluene, and mixedsolvents thereof. Alternatively, the reaction can be performed in theabsence of a solvent. The reaction temperature is usually in the rangefrom −78 to 100° C. or the boiling point of the solvent, preferably inthe range from 0 to 100° C.

Synthesis of Compound (8)

A compound (8) can be obtained by reacting compound (7) with a compound(B) in the presence of a base. Examples of the base used can includeorganic bases such as triethylamine, diisopropylethylamine,4-dimethylaminopyridine, N-methylmorpholine, pyridine, and 2,6-lutidine,and diazabicyclo[5.4.0]undec-7-ene, and inorganic bases such aspotassium carbonate, sodium carbonate, and sodium bicarbonate. Here, thesolvent used in the reaction is not particularly limited and examplesthereof include dichloromethane, chloroform, tetrahydrofuran, ethylacetate, toluene, and mixed solvents thereof. However, dried solventsare preferred. The reaction temperature is usually in the range from −78to 100° C. or the boiling point of the solvent, preferably in the rangefrom −10° C. to around room temperature. Moreover, in another method, acompound (8) can be obtained by reacting compound (6) with a compound(B) in the presence of a condensing agent. Here, examples of thecondensing agent used can include N,N′-dicyclohexylcarbodiimide and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The solvent used in thereaction is not particularly limited and examples thereof includedichloromethane, dimethylformamide, tetrahydrofuran, ethyl acetate, andmixed solvents thereof. The reaction temperature is usually in the rangefrom −78 to 100° C. or the boiling point of the solvent, preferably inthe range from 0 to 50° C. Moreover, a base such as triethylamine,diisopropylethylamine, N-methylmorpholine, or 4-dimethylaminopyridinecan be added, if necessary. Furthermore, 1-hydroxybenzotriazole,N-hydroxysuccinimide, or the like may be added as a reactionaccelerator.

Synthesis of Compound (9)

A compound (9) can be obtained by performing deprotection under thereaction conditions used in the method for producing compound (6) above.

Synthesis of Compound (1)

A compound (1) can be obtained from compound (9) and a compound (C)under reaction conditions described above in another method forproducing compound (8) above using a condensing agent. Moreover, inanother method, a compound (1) can be obtained by reacting compound (9)and a compound (C) with an acid halogenating reagent such as1-chloro-N,N,2-trimethyl-1-propenylamine in the presence of a base.Here, examples of the base used can include organic bases such astriethylamine, diisopropylethylamine, 4-dimethylaminopyridine,N-methylmorpholine, pyridine, 2,6-lutidine, anddiazabicyclo[5.4.0]undec-7-ene, and inorganic bases such as potassiumcarbonate, sodium carbonate, and sodium bicarbonate. The solvent used inthe reaction is not particularly limited and examples thereof includedichloromethane, chloroform, toluene, and mixed solvents thereof. Thereaction temperature is usually in the range from −78 to 100° C. or theboiling point of the solvent, preferably in the range from −20 to 50° C.

The compound (1) of the present invention can also be produced by thefollowing method.

[Production Method 2]

wherein R¹² means a protective group for the amino group; and R¹ to R³,R⁶, and R⁷ have the same meanings as defined above.

Examples of the protective group for the amino group include abenzyloxycarbonyl group, a tert-butyloxycarbonyl group, and a benzylgroup.

Synthesis of Compound (10)

A compound (10) can be obtained from a compound (B′) and a compound (C)under the reaction conditions used in the method for producing compound(1) (amidation of compound (9) with a compound (C)) according toProduction Method 1 above.

Synthesis of Compound (11)

Deprotection reaction conditions differ depending on the type of R¹².The synthesis may be performed under reaction conditions usually used inthis field. When R¹² is a benzyloxycarbonyl group or a benzyl group orthe like, deprotection can be performed by adding a reduction catalystsuch as palladium carbon and reacting compound (10) in a hydrogenatmosphere or in the presence of a hydrogen source such as ammoniumformate. Here, the solvent used in the reaction is not particularlylimited and examples thereof include alcohols such as methanol andethanol, tetrahydrofuran, dioxane, ethyl acetate, water, and mixedsolvents thereof. The reaction temperature is usually in the range from−78 to 100° C. or the boiling point of the solvent, preferably in therange from 0° C. to around room temperature. Moreover, when R¹² is atert-butyloxycarbonyl group or the like, deprotection can be performedby treating the compound (10) with trifluoroacetic acid or hydrochloricacid or the like. Here, the reaction solvent used in the reaction is notparticularly limited and examples thereof include dichloromethane,chloroform, tetrahydrofuran, di oxane, methanol, ethanol, water, andmixed solvents thereof. The reaction temperature is usually in the rangefrom −78 to 100° C. or the boiling point of the solvent, preferably inthe range from 0° C. to around room temperature.

Synthesis of Compound (1)

A compound (1) can be obtained from compound (11) and compound (6) orcompound (7) under the reaction conditions used in the method forproducing compound (8) (amidation of compound (B) with a compound (6) ora compound (7)) according to Production Method 1 above.

When R¹ in compound (1) of the present invention is atert-butoxycarbonyl group, a benzyloxycarbonyl group, or atrifluoroacetyl group, or the like usually used as a protective group, acompound (1) wherein R¹ is a hydrogen atom can be obtained under thereaction conditions used in the method for producing compound (11)(deprotection of R¹² in compound (10)) according to Production Method 2above or by treatment with a base such as sodium hydroxide, potassiumhydroxide, sodium carbonate, or potassium carbonate in a mixed solventof methanol or ethanol and water. Furthermore, when R¹ is a hydrogenatom, it can be converted to the defined substituent using aconventional organic chemical method. For example, R¹ can be convertedto an alkyl group or the like by treatment with a reducing agent such assodium cyanoborohydride or sodium triacetoxyborohydride in the presenceof an aldehyde derivative. Moreover, R¹ can be converted to an alkanoylgroup or an alkylsulfonyl group by reaction with an acid chloridederivative in the presence of a base such as triethylamine.

The starting material compound (3) can be synthesized according to themethod described in the document (Synlett, 1998, 623 or US2005/26916).Moreover, compound (3) can also be synthesized by the following method.

Production Method 3

wherein Z means a halogen atom such as a chlorine atom or a bromineatom; R¹³ means a trichloroethyloxysulfonyl group, a p-toluenesulfonylgroup, or the like; and Ar₁, Ar₂, and R⁵ have the same meanings asdefined above.

Synthesis of Compound (13)

A compound (13) can be obtained by treating a phosphonium salt or aphosphonic acid ester obtained from a reaction of a compound (12) and anorganic phosphorous compound such as triphenylphosphine or triethylphosphite with a base such as alkyl lithium, lithium diisopropylamide,lithium bis(trimethylsilyl)amide, sodium hydride, or potassiumtert-butoxide and then reacting the resulting product with a compound(D). Here, the solvent used in this reaction is not particularly limitedand examples thereof include diethyl ether, tetrahydrofuran, toluene,dimethylformamide, dimethyl sulfoxide, and mixed solvents thereof.However, dried solvents are preferred. The reaction temperature isusually in the range from −78 to 100° C. or the boiling point of thesolvent, preferably in the range from −78° C. to room temperature.

Synthesis of Compound (14)

A compound (14) can be synthesized according to synthesis methodsreported in various documents (e.g., Tetrahedron Lett., 2005, 46 4031;J. Am. Chem. Soc., 2002, 124, 136672; J. Am. Chem. Soc., 2001, 123,7707; Synlett, 2004, 525; and Japanese Patent Laid-Open No. 2000-72743).For example, a compound (14) can be obtained by reacting compound (13)with an alkoxysulfonamide derivative or an arylsulfonamide derivative inthe presence of a rhodium catalyst after addition of an oxidizing agentsuch as iodosobenzene acetate and a base such as magnesium oxide. Here,the solvent used in this reaction is not particularly limited andexamples thereof include diethyl ether, tetrahydrofuran, toluene,acetonitrile, and mixed solvents thereof. However, dried solvents arepreferred. The reaction temperature is usually in the range from −78 to100° C. or the boiling point of the solvent, preferably in the rangefrom −20 to 80° C.

Synthesis of Compound (15)

A compound (15) can be obtained by treating the compound (14) withammonia water. Here, the solvent used in this reaction is notparticularly limited and examples thereof include methanol, ethanol,water, tetrahydrofuran, dioxane, and mixed solvents thereof. However,organic solvents that can be mixed with water in an arbitrary ratio arepreferred. The reaction temperature is usually in the range from −78 to100° C. or the boiling point of the solvent, preferably in the rangefrom room temperature to 80° C.

Synthesis of Compound (3)

A compound (3) can be obtained by treating compound (15) withhydrochloric acid, sulfuric acid, or trifluoroacetic acid, or the like.Here, the solvent used in this reaction is not particularly limited andexamples thereof include methanol, ethanol, water, tetrahydrofuran,dioxane, and mixed solvents thereof. However, organic solvents that canbe mixed with water in an arbitrary ratio are preferred. The reactiontemperature is usually in the range from −78 to 100° C. or the boilingpoint of the solvent, preferably in the range from room temperature to80° C.

[Production Method 4]

wherein R¹¹ means a protective group for the carboxyl group; Z means ahalogen atom such as a chlorine atom or a bromine atom and Ar₁, Ar₂, andR⁵ have the same meanings as defined above.

Synthesis of Compound (16)

A compound (16) can be obtained by treating an aminonitrile formobtained from a reaction (Strecker reaction) of a compound (D) withpotassium cyanide or sodium cyanide, ammonium chloride, and ammoniawater with a mineral acid (e.g., hydrochloric acid or sulfuric acid) oran organic acid (e.g., p-toluenesulfonic acid or methanesulfonic acid).Here, the solvent used in the reaction is not particularly limited andexamples thereof include methanol, ethanol, water, tetrahydrofuran,dioxane, and mixed solvents thereof. The reaction temperature is usuallyin the range from −20 to 100° C. or the boiling point of the solvent,preferably in the range from around room temperature to 100° C.

Synthesis of Compound (17)

Each of the carboxyl group and the amino group in the compound (16) maybe protected according to a standard method. Here, the order in whichprotective groups are introduced is not particularly limited.Hereinafter, each reaction will be described. Esterification can beperformed by treatment with a halogenating reagent such as hydrogenchloride, sulfuric acid, or thionyl chloride in a lower alcoholappropriate for R¹¹, such as methanol or ethanol. The reactiontemperature is usually in the range from −78 to 100° C. or the boilingpoint of the solvent, preferably in the range from 0 to 100° C.Moreover, the tert-butoxycarbonylation of the amino group can beperformed by reaction with di-tert-butyl dicarbonate in the presence ofa base such as triethylamine, diisopropylethylamine, or4-dimethylaminopyridine. Here, the solvent used in the reaction is notparticularly limited and examples thereof include dichloromethane,chloroform, tetrahydrofuran, ethyl acetate, acetonitrile, toluene, andmixed solvents thereof. However, dried solvents are preferred. Thereaction temperature is usually in the range from −78 to 100° C. or theboiling point of the solvent, preferably in the range from 0 to 100° C.

Synthesis of Compound (18)

The ester group in compound (17) can be reduced by reaction using awater-reactive reagent such as lithium aluminum hydride at a temperatureequal to or lower than room temperature (preferably −40 to 0° C.) in anaprotic solvent such as diethyl ether, tetrahydrofuran, or dioxane togive an alcohol form. Moreover, similarly, the reaction using sodiumborohydride or the like can be performed at a temperature equal to orlower than room temperature (preferably −20° C. to around roomtemperature) using a protic solvent such as methanol, ethanol, or water,or a mixed solvent of the protic solvent and the aprotic solvent to givean alcohol form. A compound (18) can be obtained by reacting the alcoholform thus obtained with an oxidizing agent such as chromic acid[pyridinium chlorochromate (PCC), pyridinium dichromate (PDC), etc.],dimethyl sulfoxide with oxalyl chloride (Swern oxidation), dimethylsulfoxide with acetic anhydride, dimethyl sulfoxide with a sulfurtrioxide-pyridine complex,1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (Dess-Martinreagent), or 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) withhypochlorous acid. Here, the solvent used in the reaction is notparticularly limited and examples thereof include dichloromethane,chloroform, tetrahydrofuran, ethyl acetate, dimethyl sulfoxide, water,toluene, and mixed solvents thereof. The reaction temperature is usuallyin the range from −78 to 100° C. or the boiling point of the solvent,preferably in the range from −78° C. to around room temperature.

Synthesis of Compound (19)

A compound (19) can be obtained by reacting compound (18) with an aryllithium compound (Ar₁Li) or a Grignard reagent (Ar₁MgZ). Thecorresponding aryl lithium compound or Grignard reagent is acommercially available product or can be synthesized according to astandard method. The Grignard reagent can be synthesized from thecorresponding aryl halide and magnesium metal, and the organic lithiumreagent can be synthesized by halogen-metal exchange from thecorresponding aryl halide and a commercially available alkyl lithiumreagent or the like.

Here, the solvent used in the reaction is not particularly limited andexamples thereof include diethyl ether, tetrahydrofuran, dioxane,toluene, and mixed solvents thereof. However, dried solvents arepreferred. The reaction temperature is usually in the range from −78 to100° C. or the boiling point of the solvent, preferably in the rangefrom −78° C. to around room temperature.

Synthesis of Compound (20)

A compound (20) can be obtained by appropriately selecting and using theoxidation reaction conditions described in the method for producingcompound (19).

Synthesis of Compound (21)

A compound (21) can be obtained under the reaction conditions used inthe method for producing the compound (11) according to ProductionMethod 2 above, wherein R¹² is a tert-butyloxycarbonyl group.

Synthesis of Compound (22)

A compound (22) can be obtained by heating compound (21) in the presenceof sulfamide and a base such as 1,8-diazabicyclo[5.4.0]undec-7-ene.Here, the solvent used in the reaction is not particularly limited andexamples thereof include diethyl ether, tetrahydrofuran, dioxane, ethylacetate, methanol, ethanol, isopropyl alcohol, ethylene glycol,acetonitrile, toluene, and mixed solvents thereof. The reactiontemperature is usually in the range from −78 to 180° C. or the boilingpoint of the solvent, preferably in the range from 70 to 150° C.

Synthesis of Compound (23)

A compound (23) can be obtained from compound (22) by appropriatelyselecting and using the reduction reaction conditions described in themethod for producing compound (18).

Synthesis of Compound (3)

A compound (3) can be synthesized from compound (23) according to themethod described in the document (Synlett, 1998, 623-624 andUS2005/0026916). Moreover, in another method, a compound (3) can beobtained from compound (23) by hydrolyzation in the presence of a basesuch as pyridine or ethylenediamine. Here, examples of the solvent usedin the reaction include methanol, ethanol, water, tetrahydrofuran,dioxane, ethylene glycol, and mixed solvents thereof. However, organicsolvents that can be mixed with water in an arbitrary ratio arepreferred. The reaction temperature is usually in the range from 0 to180° C. or the boiling point of the solvent, preferably in the rangefrom 60 to 120° C.

The racemic compound (3) obtained by the production method can beoptically resolved according to the method described in the document(US2005/26916, Japanese Patent Laid-Open No. 2005-75754, and TetrahedronAsymmetry, 1995, 6, 3). For example, the racemic compound (3) can betreated with an optical resolving agent such as L-(+)-tartaric acid in amixed solvent of methanol or ethanol and water to give crystallinetartrate. This tartrate is treated with a base such as sodium hydroxideto give an optically active diamine (3) having the positionalconfiguration shown above.

The starting material compound (A) is a commercially available productor can be synthesized according to the method described in the document(Tetrahedron Asymmetry, 1995, 6, 2199).

The starting material compounds (B) and (C) are commercially availableproducts or can be synthesized according to methods described in theReference Examples.

The starting material compound (D) is a commercially available productor can be synthesized according to the method described in variousdocuments (e.g., J. Med. Chem. 2000, 43, 4781).

In one embodiment of the present invention, the compound of the presentinvention can be used as a p53-Mdm2 binding inhibitor and/or an Mdm2ubiquitin ligase inhibitor because it inhibits the binding of p53 withMdm2 and the ubiquitination of p53 by Mdm2.

The condition of the p53-Mdm2 binding can be examined by a methodconventionally used by those skilled in the art to examine bindingconditions between proteins (for example, immunological techniques,surface plasmon resonance techniques, etc.). Examples of methods forexamining the condition of the Mdm2-p53 binding using an immunologicaltechnique include an immuno-sedimentation method andenzyme-linked-immuno-sorbent assay (ELISA). An antibody used in suchimmunological techniques may be an anti-Mdm2 antibody and/or an anti-p53antibody that can directly detect Mdm2 and/or p53. When Mdm2 and/or p53is labeled with a tag (for example, a GST tag or a histidine tag) or thelike, an antibody suitable for labeling (for example, an anti-GSTantibody or an anti-histidine antibody) can be used. Methods forexamining the condition of the Mdm2-p53 binding using an immunologicaltechnique are described in, for example, WO2003/51359, WO2003/51360,U.S. Patent Application Publication No. 2004/259867 or 2004/259884, andWO2005/110996. Methods for examining the condition of the Mdm2-p53binding using a surface plasmon resonance technique are described in,for example, Science, vol. 303, p. 844-848, 2004.

Ubiquitin ligase activity of Mdm2 against p53 can be examined by anubiquitin ligase assay conventionally used by those skilled in the art.The ubiquitin ligase activity can be detected by, for example, comparingubiquitination of p53 by ubiquitin activation enzyme (E1), ubiquitinbinding enzyme (E2), and ubiquitin ligase (E3) (Mdm2) in the presenceand absence of a test compound (for example, refer to WO2001/75145 andWO2003/76608).

In another embodiment, the compound of the present invention can be usedas an inhibitor of suppression of the p53 transcription activity becauseit restores functions of p53 as a transcription factor that issuppressed by Mdm2 by inhibiting the binding of Mdm2 to the p53transcription activation domain. The inhibitor of suppression of the p53transcription activity can be obtained by, for example, measuring themRNA level or the protein level of a protein whose transcription isregulated by p53 (for example, p21^(Wafl/Cipl)) in the presence orabsence of a test compound by an mRNA measuring method (for example,Northern blot) or a protein measuring method (for example, Western blot)conventionally used by those skilled in the art and selecting the testcompound as an inhibitor of suppression of the p53 transcriptionactivity when the mRNA level or the protein level is increased in thepresence of the test compound as compared with that in the absence ofthe test compound. Furthermore, the inhibitor of suppression of the p53transcription activity can also be identified by a reporter assay usingthe reporter activity of a reporter gene including a p53 responsiveelement as an indicator.

In another embodiment, the compound of the present invention can be usedas a p53 degradation inhibitor because it inhibits ubiquitination of p53by Mdm2 and thereby prevents the degradation of p53 in proteasomes. Thep53 degradation inhibitor can be obtained by, for example, measuring theprotein level of p53 in the presence or absence of a test compound by aprotein measuring method (for example, Western blot) conventionally usedby those skilled in the art and selecting the test compound as a p53degradation inhibitor when the protein level is increased in thepresence of the test compound as compared with that in the absence ofthe test compound.

In another embodiment, the compound of the present invention can be usedas an anti-tumor agent because it normalizes functions of p53 as acancer-restraining gene by inhibition of the Mdm2-p53 binding and/orubiquitination of p53 by Mdm2.

Cellular growth inhibiting activity can be examined by methods fortesting growth inhibition conventionally used by those skilled in theart. The cell growth inhibition activity can be determined by, forexample, comparing the levels of cellular growth (for example, tumorcells) in the presence or absence of a test compound as described in thefollowing Test Example 2. The levels of cellular growth can be examinedby using, for example, a test system for measuring living cells.Examples of the method for measuring living cells include the[³H]-thymidine uptake test, the BrdU method, the MTT assay, and soforth.

Moreover, in vivo anti-tumor activity can be examined by methods fortesting anti-tumor activity conventionally used by those skilled in theart. The in vivo anti-tumor activity of the present invention can beconfirmed by, for example, transplanting various tumor cells to mice,rats, or the like; after confirming the engraftment of the transplantedcells, orally or intravenously administering the compound of the presentinvention to the animals; a few days or a few weeks later, comparingtumor growth in a drug-non-administered group with that in thecompound-administered group.

The compound of the present invention can be used for the treatment oftumors or cancers, for example, lung cancer, digestive system cancer,ovary cancer, uterine cancer, breast cancer, liver cancer, head/neckregion cancer, blood cancer, renal cancer, and testicular tumors, morepreferably lung cancer, breast cancer, prostate cancer, colon cancer,acute myeloid leukemia, malignant lymphoma, retinoblastoma,neuroblastoma, and sarcoma. However, the present invention is notlimited to these cancers.

A pharmaceutical composition of the present invention can contain acompound of the present invention and a pharmaceutically acceptablecarrier and can be administered as various injections such asintravenous injection, intramuscular injection, and subcutaneousinjection or by various methods such as oral administration orpercutaneous administration. Pharmaceutically acceptable carrier means apharmacologically acceptable material that is involved in transport ofthe compound of the present invention or a composition containing thecompound of present invention (for example, an excipient, a diluent, anadditive, a solvent, etc.) from a given organ to another organ.

A formulation can be prepared by selecting a suitable formulation form(for example, oral formulation or injection) depending on theadministration method and using various conventionally used methods forpreparing a formulation. Examples of oral formulations include tablets,powders, granules, capsules, pills, lozenges, solutions, syrups,elixirs, emulsions, oily or aqueous suspensions, and so forth. In oraladministration, the free compound or a salt form may be used. An aqueousformulation can be prepared by forming an acid adduct with apharmacologically acceptable acid or by forming an alkali metal saltsuch as sodium. As an injection, a stabilizer, a preservative, adissolving aid, and the like can be used in the formulation. Afterfilling a solution that may contain these aids and the like in a vessel,a formulation for use may be prepared as a solid formulation bylyophilization or the like. Furthermore, one dose may be filled in onevessel, or two or more doses may be filled in a vessel.

Examples of solid formulations include tablets, powders, granules,capsules, pills, and lozenges. These solid formulations may containpharmaceutically acceptable additives together with a compound of thepresent invention. Examples of additives include fillers, extenders,binders, disintegrating agents, dissolution promoting agents, skinwetting agents, and lubricants, and these can be selected and mixed asrequired to prepare a formulation.

Examples of liquid formulations include solutions, syrups, elixirs,emulsions, and suspensions. These liquid formulations may containpharmaceutically acceptable additives together with a compound of thepresent invention. Examples of additives include suspending agents andemulsifiers, and these are selected and mixed as required to prepare aformulation.

The compound of the present invention can be used in cancer treatment ofmammals, in particular, humans. The dose and the administration intervalcan be suitably selected depending on the site of the disease, thepatient's height, body weight, sex, or medical history, according to aphysician's judgment. When the compound of the present invention isadministered to a human, the dose range is approx. 0.01 to 500 mg/kgbody weight per day, preferably, approx 0.1 to 100 mg/kg body weight.Preferably, the compound of the present invention is administered to ahuman once a day, or the dose is divided two to four times, andadministration is repeated at an appropriate interval. Furthermore, thedaily dose may exceed the above-mentioned dose at a physician'sdiscretion, if necessary.

The compound of the present invention may be used in combination with anadditional anti-tumor agent. Examples thereof include anti-tumorantibiotics, anti-tumor plant constituents, BRMs (biological responsemodifiers), hormones, vitamins, anti-tumor antibodies, molecular targetdrugs, and other anti-tumor agents.

More specifically, examples of alkylating agents include: alkylatingagents such as nitrogen mustard, nitrogen mustard N-oxide, andchlorambucil; aziridine alkylating agents such as carboquone andthiotepa; epoxide alkylating agents such as dibromomannitol anddibromodulcitol; nitrosourea alkylating agents such as carmustine,lomustine, semustine, nimustine hydrochloride, streptozocin,chlorozotocin, and ranimustine; and busulfan, improsulfan tosylate, anddacarbazine.

Examples of various metabolic antagonists include: purine metabolicantagonists such as 6-mercaptopurine, 6-thioguanine, and thioinosine;pyrimidine metabolic antagonists such as fluorouracil, tegafur,tegafur-uracil, carmofur, doxifluridine, broxuridine, cytarabine, andenocitabine; and folic acid metabolic antagonists such as methotrexateand trimetrexate.

Examples of anti-tumor antibiotics include: anti-tumor anthracyclineantibiotics such as mitomycin C, bleomycin, peplomycin, daunorubicin,aclarubicin, doxorubicin, pirarubicin, THP-adriamycin,4′-epidoxorubicin, and epirubicin; and chromomycin A3 and actinomycin D.

Examples of anti-tumor plant constituents include: vinca alkaloids suchas vindesine, vincristine, and vinblastine; taxanes such as paclitaxeland docetaxel; and epipodophyllotoxins such as etoposide and teniposide.

Examples of BRMs include tumor necrosis factors and indomethacin.

Examples of hormones include hydrocortisone, dexamethasone,methylprednisolone, prednisolone, prasterone, betamethasone,triamcinolone, oxymetholone, nandrolone, metenolone, fosfestrol,ethinylestradiol, chlormadinone, and medroxyprogesterone.

Examples of vitamins include vitamin C and vitamin A.

Examples of anti-tumor antibodies and molecular target drugs includetrastuzumab, rituximab, cetuximab, nimotuzumab, denosumab, bevacizumab,infliximab, imatinib mesilate, gefitinib, erlotinib, sunitinib,lapatinib, and sorafenib.

Examples of other anti-tumor agents include cisplatin, carboplatin,oxaliplatin, tamoxifen, camptothecin, ifosfamide, cyclophosphamide,melphalan, L-asparaginase, aceglatone, sizofiran, picibanil,procarbazine, pipobroman, neocarzinostatin, hydroxyurea, ubenimex, andkrestin.

The present invention also includes a method for preventing and/ortreating cancer, comprising administering a compound of the presentinvention or a salt thereof.

The present invention further includes use of a compound of the presentinvention, a salt, or a solvate thereof for the manufacture of themedicament.

Hereinafter, the present invention will be specifically explained withreference to the Examples. However, the present invention is not limitedto these examples, and they should not be construed in any limitativeway. Furthermore, reagents, solvents, and starting materials in thespecification can be readily obtained from commercially available supplysources unless otherwise specified.

EXAMPLES Example 1

Step 1:7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

Thionyl chloride (0.50 ml) and dimethylformamide (2 drops) were added toa 1,2-dichloroethane (10 ml) suspension of the compound (470 mg, 1.00mmol) obtained in Step 13 of Reference Example 1, the resulting mixturewas stirred under heating at 70° C. for 1 hour and then the solvent wasconcentrated under reduced pressure. The residue obtained was dissolvedin tetrahydrofuran (10 ml) and added dropwise to a tetrahydrofuran (10ml) solution of the compound (380 mg, 1.20 mmol) obtained in Step 2 ofReference Example 3 and triethylamine (0.42 ml, 3.00 mmol) under icecooling and the resulting mixture was stirred at room temperature for 20hours. The reaction mixture was diluted with ethyl acetate and theorganic layer was washed with saturated aqueous sodium bicarbonatesolution and saturated brine and dried over anhydrous sodium sulfate.The solvent was evaporated under reduced pressure and the residueobtained was purified by NH-silica gel column chromatography[n-hexane:ethyl acetate=1:5 (v/v)] to give the title compound (399 mg,51%) as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.93 (3H, d, J=7.1 Hz), 0.93 (3H,d, J=7.1 Hz), 0.98-1.12 (4H, m), 1.77 (3H, s), 2.05-2.15 (1H, m),2.54-2.60 (1H, m), 2.61-2.67 (1H, m), 3.49-3.57 (2H, m), 3.63-3.83 (5H,m), 3.87-3.97 (1H, m), 5.06-5.09 (1H, m), 5.36 (1H, d, J=53.7 Hz), 5.51(1H, s), 6.73-6.77 (1H, m), 6.91-6.97 (1H, m), 7.17 (1H, d, J=8.3 Hz),7.34 (1H, t, J=7.9 Hz), 7.65 (1H, dd, J=8.3, 2.7 Hz), 8.26 (1H, d, J=2.4Hz).

MS (ESI) m/z: 771 [(M+H)⁺].

Step 2:7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-4,7-diazaspiro[2.5]octane

The compound (390 mg, 0.51 mmol) obtained in Step 1 above was dissolvedin 10% aqueous methanol (8 ml), potassium carbonate (400 mg, 2.89 mmol)was added and the resulting mixture was stirred under heating at 40° C.for 2 hours. The solvent was concentrated under reduced pressure andthen the residue obtained was diluted with ethyl acetate, washed withsaturated brine, and then dried over anhydrous sodium sulfate. Thesolvent was evaporated under reduced pressure and the residue obtainedwas purified by silica gel column chromatography[chloroform:methanol=15:1 (v/v)] to give the title compound (190 mg,55%) as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.44-0.52 (4H, m), 0.93 (3H, d,J=6.8 Hz), 0.94 (3H, d, J=6.8 Hz), 1.78 (3H, s), 2.05-2.15 (1H, m),2.56-2.64 (1H, m), 2.61-2.69 (1H, m), 2.73-2.79 (2H, m), 3.31-3.39 (2H,m), 3.43-3.50 (2H, m), 3.74 (1H, dd, J=35.8, 12.1 Hz), 3.91 (1H, dd,J=19.2, 12.6 Hz), 5.02-5.06 (1H, m), 5.35 (1H, d, J=53.7 Hz), 5.52 (1H,s), 6.73-6.78 (1H, m), 6.92-6.99 (1H, m), 7.18 (1H, dd, J=8.3, 0.7 Hz),7.35 (1H, t, J=8.1 Hz), 7.66 (1H, dd, J=8.3, 2.7 Hz), 8.27 (1H, d, J=2.4Hz).

MS (ESI) m/z: 675 [(M+H)⁺].

Anal. Calcd. for C₃₂H₃₄Cl₂F₂N₆O₂S.0.25 H₂O: C, 56.51; H, 5.11; N, 12.36;F, 5.59; Cl, 10.43; S, 4.71.

Found: C, 56.33; H, 5.10; N, 12.20; F, 5.55; Cl, 10.21; S, 4.73.

Example 2

Step 1:(6S)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

Triethylamine (0.12 ml, 0.86 mmol),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (100 mg,0.52 mmol), and 1-hydroxybenzotriazole (53 mg, 0.43 mmol) were added toa dimethylformamide (3 ml) solution of the compound (200 mg, 0.43 mmol)obtained in Step 13 of Reference Example 1 and the compound (159 mg,0.47 mmol) obtained in Step 2 of Reference Example 5 and the resultingmixture was stirred at room temperature for 24 hours. The reactionmixture was diluted with ethyl acetate, washed with saturated aqueoussodium bicarbonate solution, water, and saturated brine, and then driedover anhydrous sodium sulfate. The solvent was evaporated under reducedpressure and the residue obtained was purified by silica gel columnchromatography [n-hexane:ethyl acetate=1:1 (v/v)] to give the titlecompound (286 mg, 85%) as a pale yellow solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.72-0.74 (2H, m), 0.92 (3H, d,J=7.1 Hz), 0.96 (3H, d, J=7.1 Hz), 1.12 (3H, brd, J=6.1 Hz), 1.30 (1H,m), 1.49 (1H, m), 1.78 (3H, s), 2.17 (1H, m), 2.58-2.65 (2H, m),3.54-3.57 (3H, m), 3.78-3.92 (3H, m), 4.69 (1H, m), 5.01 (1H, m), 5.39(1H, d, J=53.1 Hz), 5.51 (1H, s), 6.76 (1H, d, J=8.3 Hz), 6.95 (1H, d,J=8.3 Hz), 7.17 (1H, d, J=8.3 Hz), 7.34 (1H, t, J=8.3 Hz), 7.65 (1H, dd,J=8.3, 2.7 Hz), 8.26 (1H, d, J=2.7 Hz).

MS (ESI) m/z: 785 [(M+H)⁺].

Step 2:(6S)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-6-methyl-4,7-diazaspiro[2.5]octane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.35-0.41 (2H, m), 0.57-0.59 (2H,m), 0.92 (3H, d, J=7.1 Hz), 0.95 (3H, d, J=7.1 Hz), 1.21-1.22 (3H, brd,J=5.6 Hz), 1.28 (1H, m), 1.78 (3H, s), 2.14 (1H, m), 2.55-2.66 (3H, m),3.21-3.44 (2H, m), 3.75 (1H, ddd, J=36.6, 12.8, 2.9 Hz), 3.92 (1H, dd,J=18.2, 12.8 Hz), 4.33 (1H, m), 4.97 (1H, m), 5.36 (1H, d, J=52.9 Hz),5.50 (1H, s), 6.75 (1H, d, J=8.3 Hz), 6.95 (1H, d, J=8.3 Hz), 7.17 (1H,dd, J=8.3, 0.6 Hz), 7.34 (1H, t, J=8.3 Hz), 7.65 (1H, dd, J=8.3, 2.4Hz), 8.26 (1H, dd, J=2.4, 0.6 Hz).

MS (ESI) m/z: 689 [(M+H)⁺].

Anal. Calcd. for C₃₃H₃₆Cl₂F₂N₆O₂S.1.25 H₂O: C, 55.65; H, 5.45; N, 11.80.

Found: C, 55.25; H, 5.23; N, 11.32.

Example 3

Step 1:(6R)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 13 of Reference Example 1 was reacted inthe same way as in Step 1 of Example 2 using the compound obtained inStep 2 of Reference Example 7 instead of the compound obtained in Step 2of Reference Example 5 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.71-0.73 (1H, m), 0.89-0.92 (1H,m), 0.93 (6H, d, J=7.1 Hz), 1.10-1.13 (3H, m), 1.30-1.32 (1H, m),1.47-1.49 (1H, m), 1.77 (3H, s), 2.06-2.08 (1H, m), 2.61-2.68 (1H, m),3.51-3.54 (3H, m), 3.71-3.81 (1H, m), 3.90-3.95 (2H, m), 4.61 (1H, brs),5.04 (1H, t, J=8.1 Hz), 5.34 (1H, d, J=54.0 Hz), 5.50 (1H, s), 6.74 (1H,d, J=7.8 Hz), 6.93 (1H, d, J=7.8 Hz), 7.16 (1H, dd, J=8.3, 0.7 Hz), 7.33(1H, t, J=7.8 Hz), 7.64 (1H, dd, J=8.3, 2.4 Hz), 8.25 (1H, t, J=1.3 Hz).

Step 2:(6R)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-6-methyl-4,7-diazaspiro[2.5]octane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.35-0.37 (2H, m), 0.55-0.57 (2H,m), 0.92 (3H, d, J=7.1 Hz), 0.93 (3H, d, J=7.1 Hz), 1.26 (3H, brs), 1.76(3H, s), 1.99-2.02 (1H, m), 2.32-2.34 (1H, m), 2.61-2.68 (3H, m),2.86-2.92 (1H, m), 3.30-3.32 (1H, m), 3.72 (1H, dd, J=36.3, 11.9 Hz),3.90 (1H, dd, J=19.9, 12.8 Hz), 4.17-4.20 (1H, m), 5.00 (1H, t, J=8.0Hz), 5.32 (1H, d, J=53.1 Hz), 5.48 (1H, s), 6.73 (1H, d, J=8.3 Hz), 6.92(1H, d, J=8.3 Hz), 7.15 (1H, d, J=8.3 Hz), 7.31 (1H, t, J=7.9 Hz), 7.63(1H, dd, J=8.3, 2.7 Hz), 8.24 (1H, d, J=2.7 Hz).

MS (ESI) m/z: 689 [(M+H)⁺].

Anal. Calcd. for C₃₃H₃₆Cl₂F₂N₆O₂S.1.25 H₂O: C, 55.65; H, 5.45; N, 11.80.

Found: C, 55.26; H, 5.19; N, 11.85.

Example 4

Step 1:(6S)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-6-ethyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 13 of Reference Example 1 was reacted inthe same way as in Step 1 of Example 2 using the compound obtained inStep 2 of Reference Example 9 instead of the compound obtained in Step 2of Reference Example 5 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.67-0.74 (2H, m), 0.80-0.85 (3H,m), 0.91 (3H, d, J=7.1 Hz), 0.94 (3H, d, J=6.8 Hz), 1.25-1.33 (1H, m),1.43-1.53 (3H, m), 1.77 (3H, s), 2.17-2.27 (2H, m), 2.55-2.63 (1H, m),3.37-3.97 (6H, m), 4.06-4.13 (1H, m), 4.49-4.53 (1H, m), 5.40 (1H, d,J=52.2 Hz), 5.51 (1H, s), 6.72-6.78 (1H, m), 6.92-6.99 (1H, m), 7.17(1H, d, J=8.3 Hz), 7.34 (1H, t, J=8.1 Hz), 7.65 (1H, dd, J=8.4, 2.6 Hz),8.26 (1H, d, J=2.2 Hz).

MS (ESI) m/z: 799 [(M+H)⁺].

Step 2:(6S)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-6-ethyl-4,7-diazaspiro[2.5]octane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.31-0.39 (2H, m), 0.50-0.58 (2H,m), 0.78-0.84 (3H, m), 0.91 (3H, d, J=7.3 Hz), 0.93 (3H, d, J=7.1 Hz),1.65-1.73 (2H, m), 1.77 (3H, s), 2.09-2.22 (1H, m), 2.30-2.36 (1H, m),2.56-2.63 (1H, m), 2.72-2.77 (2H, m), 3.46-3.58 (2H, m), 3.70-3.95 (2H,m), 4.12-4.20 (1H, m), 4.89-4.95 (1H, m), 5.37 (1H, d, J=53.2 Hz), 5.50(1H, s), 6.72-6.79 (1H, m), 6.91-6.99 (1H, m), 7.17 (1H, d, J=8.3 Hz),7.34 (1H, t, J=7.9 Hz), 7.65 (1H, dd, J=8.4, 2.6 Hz), 8.26 (1H, d, J=2.4Hz).

MS (ESI) m/z: 703 [(M+H)⁺].

Anal. Calcd. for C₃₄H₃₈Cl₂F₂N₆O₂S.0.25 H₂O: C, 57.66; H, 5.48; N, 11.87;F, 5.37; Cl, 10.01; S, 4.53.

Found: C, 57.56; H, 5.49; N, 11.74; F, 5.32; Cl, 9.78; S, 4.49.

Example 5

(6R)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-6-ethyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 13 of Reference Example 1 was reacted inthe same way as in Step 1 of Example 2 using the compound obtained inStep 2 of Reference Example 11 instead of the compound obtained in Step2 of Reference Example 5 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.69-0.75 (2H, m), 0.79-0.86 (3H,m), 0.94 (6H, d, J=7.1 Hz), 1.05-1.14 (1H, m), 1.26-1.33 (1H, m),1.45-1.53 (3H, m), 1.78 (3H, s), 1.92-2.12 (1H, m), 2.60-2.70 (1H, m),3.27-3.49 (2H, m), 3.64-3.84 (2H, m), 3.89-3.99 (1H, m), 4.14-4.25 (1H,m), 4.58-4.64 (1H, m), 5.06-5.12 (1H, m), 5.35 (1H, d, J=55.7 Hz), 5.52(1H, s), 6.74-6.79 (1H, m), 6.92-6.98 (1H, m), 7.18 (1H, d, J=8.3 Hz),7.35 (1H, t, J=7.9 Hz), 7.66 (1H, dd, J=8.2, 2.3 Hz), 8.27 (1H, d, J=2.2Hz).

MS (ESI) m/z: 799 [(M+H)⁺].

Anal. Calcd. for C₃₆H₃₇Cl₂F₅N₆O₃S.0.5 H₂O: C, 53.47; H, 4.74; N, 10.39;F, 11.75; Cl, 8.77; S, 3.97.

Found: C, 52.97; H, 4.52; N, 10.37; F, 12.45; Cl, 7.90; S, 3.94.

Example 6

(6R)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-6-ethyl-4,7-diazaspiro[2.5]octane

The compound obtained in Example 5 was reacted in the same way as inStep 2 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 70° C.) δ: 0.25-0.38 (2H, m), 0.49-0.55 (1H,m), 0.57-0.64 (1H, m), 0.78-0.98 (3H, m), 0.93 (6H, d, J=7.1 Hz),1.62-1.72 (1H, m), 1.77 (3H, s), 1.86-2.13 (1H, m), 2.40-2.45 (1H, m),2.59-2.67 (1H, m), 2.69-2.84 (3H, m), 3.49 (1H, dd, J=13.3, 6.5 Hz),3.63-3.82 (2H, m), 3.86-3.97 (1H, m), 4.17-4.24 (1H, m), 4.98-5.06 (1H,m), 5.33 (1H, dd, J=53.1, 20.1 Hz), 5.53 (1H, s), 6.71-6.75 (1H, m),6.93-6.99 (1H, m), 7.19 (1H, d, J=8.3 Hz), 7.35 (1H, t, J=7.4 Hz), 7.66(1H, dd, J=8.4, 2.3 Hz), 8.26 (1H, d, J=2.2 Hz).

MS (ESI) m/z: 703 [(M+H)⁺].

Anal. Calcd. for C₃₄H₃₈Cl₂F₂N₆O₂S.0.25 H₂O: C, 57.66; H, 5.48; N, 11.87;F, 5.36; Cl, 10.01; S, 4.53.

Found: C, 57.71; H, 5.40; N, 11.65; F, 4.99; Cl, 9.43; S, 4.43.

Example 7

(6R)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-6-isobutyl-4,7-diazaspiro[2.5]octane

The compound obtained in Step 13 of Reference Example 1 was reacted inthe same way as in Step 1 of Example 2 using the compound obtained inStep 2 of Reference Example 13 instead of the compound obtained in Step2 of Reference Example 5 and then reacted in the same way as in Step 2of Example 1 to give the title compound as a pale yellow solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.22-0.76 (4H, m), 0.83-1.03 (13H,m), 1.40-1.56 (1H, m), 1.59-1.76 (1H, m), 1.79 (3H, s), 1.81-3.04 (7H,m), 3.06-3.22 (1H, m), 3.44-3.63 (1H, m), 3.64-3.85 (1H, m), 3.92 (1H,dd, J=19.9, 12.8 Hz), 4.92-5.10 (1H, m), 5.22-5.46 (1H, m), 5.51 (1H,s), 6.76 (1H, d, J=8.1 Hz), 6.95 (1H, d, J=8.1 Hz), 7.18 (1H, d, J=8.3Hz), 7.34 (1H, t, J=8.1 Hz), 7.66 (1H, dd, J=8.3, 2.4 Hz), 8.27 (1H, d,J=2.4 Hz).

MS (ESI) m/z: 731 [(M+H)⁺].

Anal. Calcd. for C₃₆H₄₂Cl₂F₂N₆O₂S.0.5 H₂O: C, 58.37; H, 5.85; N, 11.35.

Found: C, 58.42; H, 5.77; N, 11.17.

Example 8

4-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-4,9-diazaspiro[2.2.2.2]decane

The compound obtained in Step 13 of Reference Example 1 was reacted inthe same way as in Step 1 of Example 2 using the compound obtained inStep 2 of Reference Example 15 instead of the compound obtained in Step2 of Reference Example 5 and then reacted in the same way as in Step 2of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.60 (4H, d, J=5.1 Hz), 0.87 (1H,t, J=7.0 Hz), 0.94 (6H, t, J=6.8 Hz), 1.08-1.23 (5H, m), 1.79 (3H, s),2.13 (1H, dd, J=36.3, 6.2 Hz), 2.59-2.74 (3H, m), 3.58 (2H, dd, J=34.9,12.9 Hz), 3.79-4.00 (2H, m), 5.21 (1H, s), 5.39 (1H, d, J=53.5 Hz), 5.51(1H, s), 6.76 (1H, d, J=7.8 Hz), 6.95 (1H, d, J=9.3 Hz), 7.17 (1H, d,J=8.3 Hz), 7.34 (1H, t, J=7.9 Hz), 7.65 (1H, dd, J=8.4, 2.3 Hz), 8.26(1H, d, J=2.4 Hz).

MS (ESI) m/z: 701 [(M+H)⁺].

Anal. Calcd. for C₃₄H₃₆Cl₂F₂N₆O₂S.1.75 H₂O: C, 55.69; H, 5.43; N, 11.46.

Found: C, 55.58; H, 5.32; N, 11.01.

Example 9

Step 1:(6S)-7-(1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4,4-difluoro-L-prolyl)-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 13 of Reference Example 1 was reacted inthe same way as in Step 1 of Example 2 using the compound obtained inStep 2 of Reference Example 16 instead of the compound obtained in Step2 of Reference Example 5 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.73 (2H, brs), 0.90-0.95 (6H, m),1.12 (3H, brs), 1.25-1.31 (1H, m), 1.48-1.50 (1H, m), 1.77 (3H, s),2.31-2.45 (2H, m), 2.58-2.65 (1H, m), 2.91-2.96 (2H, m), 3.38-3.58 (3H,m), 3.93-4.19 (2H, m), 4.65 (1H, brs), 5.10 (1H, br), 5.50 (1H, s), 6.77(1H, d, J=7.5 Hz), 6.96 (1H, d, J=10.0 Hz), 7.17 (1H, d, J=8.2 Hz), 7.32(1H, t, J=8.0 Hz), 7.64 (1H, dd, J=8.2, 2.2 Hz), 8.25 (1H, d, J=2.2 Hz).

MS (ESI) m/z: 803 [(M+H)⁺].

Step 2:(6S)-7-(1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4,4-difluoro-L-prolyl)-6-methyl-4,7-diazaspiro[2.5]octane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.39 (2H, t, J=7.7 Hz), 0.57 (2H,t, J=7.7 Hz), 0.92 (6H, t, J=7.6 Hz), 1.18-1.29 (5H, m), 1.77 (3H, s),2.28-2.41 (1H, m), 2.62-2.68 (2H, m), 3.39 (3H, br), 3.90-4.01 (1H, m),4.09 (1H, t, J=12.5 Hz), 4.28 (1H, br), 5.10 (1H, br), 5.50 (1H, s),6.76 (1H, d, J=7.8 Hz), 6.96 (1H, d, J=9.5 Hz), 7.16 (1H, dd, J=8.3, 0.5Hz), 7.32 (1H, t, J=8.0 Hz), 7.64 (1H, dd, J=8.3, 2.5 Hz), 8.24 (1H, d,J=2.0 Hz).

MS (ESI) m/z: 707 [(M+H)⁺].

Anal. Calcd. for C₃₃H₃₅Cl₂F₃N₆O₂S.0.5 H₂O: C, 55.31; H, 5.06; N, 11.73.

Found: C, 55.69; H, 5.28; N, 11.52.

Example 10

Step 1:(6R)-7-(1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4,4-difluoro-L-prolyl)-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 13 of Reference Example 1 was reacted inthe same way as in Step 1 of Example 2 using the compound obtained inStep 2 of Reference Example 17 instead of the compound obtained in Step2 of Reference Example 5 to give the title compound as a colorlesssolid.

MS (ESI) m/z: 803 [(M+H)⁺].

Step 2:(6R)-7-(1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4,4-difluoro-L-prolyl)-6-methyl-4,7-diazaspiro[2.5]octane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.38 (1H, br), 0.58 (3H, br), 0.93(6H, t, J=7.3 Hz), 1.23-1.28 (3H, m), 1.78 (3H, s), 2.32 (1H, br),2.46-2.50 (2H, m), 2.66-2.72 (2H, m), 2.93-3.01 (3H, m), 3.94-4.12 (2H,m), 5.13 (1H, dd, J=9.6, 5.1 Hz), 5.51 (1H, s), 6.76 (1H, d, J=8.0 Hz),6.95 (1H, d, J=9.8 Hz), 7.16 (1H, dd, J=8.0, 0.5 Hz), 7.32 (1H, t, J=8.0Hz), 7.64 (1H, dd, J=8.3, 2.5 Hz), 8.24 (1H, d, J=2.5 Hz).

MS (ESI) m/z: 707 [(M+H)⁺].

Anal. Calcd. for C₃₃H₃₅O₂F₃N₆O₂S.0.5 H₂O: C, 55.31; H, 5.06; N, 11.73.

Found: C, 55.49; H, 5.18; N, 11.47.

Example 11

Step 1:(6R)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 13 of Reference Example 1 was reacted inthe same way as in Step 1 of Example 2 using the compound obtained inReference Example 19 instead of the compound obtained in Step 2 ofReference Example 5 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: −0.01 (3H, s), 0.00 (3H, s),0.63-0.76 (2H, m), 0.83 (9H, s), 0.88 (3H, d, J=7.1 Hz), 0.92 (3H, d,J=7.1 Hz), 1.23-1.30 (1H, m), 1.38-1.47 (1H, m), 1.74 (3H, s), 2.07-2.29(2H, m), 2.52-2.62 (1H, m), 3.47-3.96 (7H, m), 4.10-4.22 (1H, m),4.41-4.53 (1H, m), 4.92-5.04 (1H, m), 5.37 (1H, d, J=53.0 Hz), 5.49 (1H,s), 6.70-6.74 (1H, m), 6.89-6.95 (1H, m), 7.14 (1H, d, J=8.5 Hz), 7.31(1H, t, J=8.1 Hz), 7.62 (1H, dd, J=8.3, 2.4 Hz), 8.23 (1H, d, J=2.4 Hz).

MS (ESI) m/z: 915 [(M+1)⁺].

Step 2:{(6R)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-4,7-diazaspiro[2.5]oct-6-yl}methanol

Tetra-n-butylammonium fluoride (1 M tetrahydrofuran solution) (0.42 ml,0.42 mmol) was added to a tetrahydrofuran (4 ml) solution of thecompound (255 mg, 0.28 mmol) obtained in Step 1 above and the resultingmixture was stirred at room temperature for 3 hours. The solvent wasconcentrated under reduced pressure, then the residue obtained wasdiluted with ethyl acetate and the organic layer was washed withsaturated aqueous sodium bicarbonate solution and saturated brine anddried over anhydrous sodium sulfate. The solvent was evaporated underreduced pressure and the residue obtained was purified by NH-silica gelcolumn chromatography [chloroform:methanol=40:1 (v/v)] to give the titlecompound (102 mg, 52%) as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.33-0.41 (2H, m), 0.51-0.59 (2H,m), 0.92 (3H, d, J=7.1 Hz), 0.95 (3H, d, J=7.1 Hz), 1.78 (3H, s),2.11-2.31 (2H, m), 2.56-2.64 (1H, m), 3.50-3.97 (6H, m), 4.09-4.18 (1H,m), 4.47-4.64 (1H, m), 4.83-4.94 (1H, m), 5.08-5.19 (1H, m), 5.37 (1H,d, J=53.2 Hz), 5.51 (1H, s), 6.72-6.78 (1H, m), 6.93-7.00 (1H, m), 7.18(1H, d, J=8.3 Hz), 7.35 (1H, t, J=7.9 Hz), 7.66 (1H, dq, J=8.4, 1.2 Hz),8.27 (1H, d, J=2.2 Hz).

MS (ESI) m/z: 705 [(M+H)⁺].

Anal. Calcd. for C₃₃H₃₆Cl₂F₂N₆O₃S.0.5 H₂O: C, 55.46; H, 5.22; N, 11.60;F, 5.32; Cl, 9.92; S, 4.49.

Found: C, 55.42; H, 5.24; N, 11.73; F, 5.28; Cl, 9.77; S, 4.43.

Example 12

Step 1:(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 13 of Reference Example 1 was reacted inthe same way as in Step 1 of Example 2 using the compound obtained inReference Example 20 instead of the compound obtained in Step 2 ofReference Example 5 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.00 (3H, s), 0.01 (3H, s),0.67-0.75 (1H, m), 0.82-0.88 (1H, m), 0.84 (9H, s), 0.87-0.94 (6H, m),1.26-1.36 (1H, m), 1.39-1.47 (1H, m), 1.74 (3H, s), 1.90-2.10 (2H, m),2.56-2.65 (1H, m), 3.40-3.95 (7H, m), 4.16-4.29 (1H, m), 4.44-4.56 (1H,m), 4.99-5.12 (1H, m), 5.32 (1H, d, J=54.2 Hz), 5.48 (1H, s), 6.69-6.74(1H, m), 6.88-6.93 (1H, m), 7.14 (1H, d, J=8.3 Hz), 7.30 (1H, t, J=8.1Hz), 7.62 (1H, dd, J=8.3, 2.7 Hz), 8.23 (1H, d, J=2.2 Hz).

MS (ESI) m/z: 915 [(M+H)⁺].

Step 2:{(6S)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-4,7-diazaspiro[2.5]oct-6-yl}methanol

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Example 11 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.25-0.40 (2H, m), 0.48-0.66 (2H,m), 0.93 (3H, d, J=7.8 Hz), 0.94 (3H, d, J=7.1 Hz), 1.78 (3H, s),1.97-2.15 (2H, m), 2.64-2.69 (1H, m), 2.75-2.85 (2H, m), 3.45-3.53 (1H,m), 3.65-3.97 (4H, m), 4.20-4.30 (1H, m), 4.41-4.51 (1H, m), 5.06 (1H,t, J=8.1 Hz), 5.34 (1H, d, J=57.1 Hz), 5.51 (1H, s), 6.71-6.77 (1H, m),6.91-6.98 (1H, m), 7.18 (1H, d, J=8.3 Hz), 7.34 (1H, t, J=8.1 Hz), 7.66(1H, dd, J=8.3, 2.4 Hz), 8.26 (1H, d, J=2.4 Hz).

MS (ESI) m/z: 705 [(M+H)⁺].

Anal. Calcd. for C₃₃H₃₆Cl₂F₂N₆O₃S.0.75 H₂O: C, 55.11; H, 5.25; N, 11.60;F, 5.28; Cl, 9.86; S, 4.46.

Found: C, 55.00; H, 5.14; N, 11.62; F, 5.27; Cl, 9.75; S, 4.45.

Example 13

Step 1: tert-butyl(6R)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-4,7-diazaspiro[2.5]octane-4-carboxylate

The compound obtained in Step 13 of Reference Example 1 was reacted inthe same way as in Step 1 of Example 1 using the compound obtained inReference Example 22 instead of the compound obtained in Step 2 ofReference Example 3 to give the title compound as a pale yellow solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.05 (6H, s), 0.46-0.54 (2H, m),0.88 (9H, s), 0.92 (3H, d, J=7.1 Hz), 0.95 (3H, d, J=6.8 Hz), 1.03-1.10(1H, m), 1.28-1.35 (1H, m), 1.42 (9H, s), 1.78 (3H, s), 2.17-2.36 (2H,m), 2.57-2.63 (1H, m), 3.14-3.26 (2H, m), 3.48-3.77 (2H, m), 3.84-3.99(4H, m), 4.37-4.48 (1H, m), 4.99-5.13 (1H, m), 5.39 (1H, d, J=52.5 Hz),5.52 (1H, s), 6.74-6.78 (1H, m), 6.93-6.99 (1H, m), 7.17 (1H, d, J=8.3Hz), 7.34 (1H, t, J=8.1 Hz), 7.66 (1H, dd, J=8.3, 2.4 Hz), 8.26 (1H, d,J=2.0 Hz).

MS (ESI) m/z: 920 [(M+1)]⁺.

Step 2: tert-butyl(6R)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-6-(hydroxymethyl)-4,7-diazaspiro[2.5]octane-4-carboxylate

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Example 11 to give the title compound as a light brown solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.43-0.53 (2H, m), 0.92 (3H, d,J=5.1 Hz), 0.96 (3H, d, J=6.8 Hz), 1.01-1.08 (1H, m), 1.29-1.34 (1H, m),1.42 (9H, s), 1.78 (3H, s), 2.17-2.31 (2H, m), 2.56-2.61 (1H, m),3.17-3.24 (1H, m), 3.46-3.54 (2H, m), 3.56-3.62 (1H, m), 3.70-3.77 (1H,m), 3.84-4.01 (2H, m), 4.32-4.36 (1H, m), 4.64-4.70 (1H, m), 5.09-5.15(1H, m), 5.38 (1H, d, J=51.3 Hz), 5.51 (1H, s), 6.72-6.79 (1H, m),6.93-6.99 (1H, m), 7.18 (1H, d, J=7.8 Hz), 7.31-7.38 (1H, m), 7.62-7.68(1H, m), 8.24-8.29 (1H, m).

MS (ESI) m/z: 805 [(M+1)]⁺.

Step 3:(6R)-4-(tert-butoxycarbonyl)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-4,7-diazaspiro[2.5]octane-6-carboxylicacid

The compound (100 mg, 0.12 mmol) obtained in Step 2 above was dissolvedin a mixed solvent of acetonitrile (1.5 ml) and water (1.5 ml),iodobenzene diacetate (88 mg, 0.26 mmol) and2,2,6,6-tetramethylpiperidine 1-oxyl (20 mg, 0.12 mmol) were added andthe resulting mixture was stirred at room temperature for 4 hours.Aqueous sodium thiosulfate solution (4 ml) was added, followed byextraction with ethyl acetate. The organic layer was washed withsaturated brine and dried over anhydrous sodium sulfate. The solvent wasevaporated under reduced pressure and the residue obtained was purifiedby silica gel chromatography [chloroform:methanol=40:1→10:1 (v/v)] togive the title compound (58 mg, 59%) as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.49-0.61 (2H, m), 0.92 (3H, d,J=7.1 Hz), 0.95 (3H, d, J=7.1 Hz), 1.07-1.16 (1H, m), 1.27-1.34 (1H, m),1.39 (9H, s), 1.78 (3H, s), 2.07-2.20 (2H, m), 2.59-2.65 (1H, m),3.16-3.22 (1H, m), 3.33-3.41 (1H, m), 3.74 (1H, dd, J=36.4, 11.0 Hz),3.87-4.04 (2H, m), 4.35-4.48 (1H, m), 4.67-4.74 (1H, m), 5.03-5.10 (1H,m), 5.35 (1H, d, J=53.0 Hz), 5.51 (1H, s), 6.74-6.78 (1H, m), 6.93-6.99(1H, m), 7.18 (1H, d, J=8.3 Hz), 7.35 (1H, t, J=8.1 Hz), 7.66 (1H, dd,J=8.4, 2.6 Hz), 8.26 (1H, d, J=2.2 Hz).

MS (ESI) m/z: 819 [(M+1)]⁺.

Step 4:(6R)-7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-4,7-diazaspiro[2.5]octane-6-carboxylicacid dihydrochloride

The compound obtained in Step 3 above was reacted in the same way as inStep 2 of Reference Example 3 to give the title compound as a paleyellow solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.85-0.90 (1H, m), 0.92 (3H, d,J=6.8 Hz), 0.98-1.02 (1H, m), 1.03 (3H, d, J=7.1 Hz), 1.10-1.19 (2H, m),2.00 (3H, s), 2.17-2.23 (1H, m), 2.26-2.33 (1H, m), 2.61-2.68 (1H, m),3.29-3.35 (2H, m), 3.68-3.74 (1H, m), 3.77-3.86 (1H, m), 4.02 (1H, dd,J=19.8, 13.4 Hz), 5.17-5.31 (2H, m), 5.42 (1H, d, J=53.5 Hz), 5.52-5.56(1H, m), 5.93 (1H, s), 6.83-6.90 (1H, m), 7.11-7.17 (1H, m), 7.27 (1H,d, J=8.5 Hz), 7.40 (1H, t, J=7.9 Hz), 7.68 (1H, dd, J=8.4, 2.6 Hz), 8.29(1H, d, J=2.2 Hz).

MS (ESI) m/z: 719 [(M+1)]⁺.

The compound obtained in Step 13 of Reference Example 1 and the compoundobtained in Reference Example 23 were reacted in the same way as inExample 13 above to give the compound in the following table.

TABLE 13 Example 14

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.92 (3H, d, J = 6.1 Hz), 0.98-1.03(2H, m), 1.04 (3H, d, J = 6.1 Hz), 1.16-1.23 (2H, m), 2.05 (3H, s),2.05-2.10 (1H, m), 2.13-2.22 (1H, m), 2.63-2.72 (1H, m), 3.27-3.35 (1H,m), 3.64-3.80 (4H, m), 4.00-4.10 (1H, m), 5.25-5.31 (2H, m), 5.39 (1H,d, J = 53.2 Hz), 6.04 (1H, s), 6.87-6.93 (1H, m), 7.14-7.21 (1H, m),7.28 (1H, d, J = 8.3 Hz), 7.40 (1H, t, J = 7.9 Hz), 7.69 (1H, dd, J =8.5, 2.7 Hz), 8.30 (1H, d, J = 2.2 Hz). MS (ESI) m/z: 719 [(M + 1)]⁺.

Example 15

7-[(4R)-1-{[(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazol-2-yl]carbonyl}-4-fluoro-L-prolyl]-4-methyl-4,7-diazaspiro[2.5]octane

Sodium triacetoxyborohydride (267 mg, 1.26 mmol) was added to a1,4-dioxane (10 ml) solution of the compound (340 mg, 0.503 mmol)obtained in Step 2 of Example 1 and 37% aqueous paraformaldehydesolution (0.41 ml, 5.05 mmol) at room temperature and the resultingmixture was stirred for 17 hours. Saturated aqueous sodium bicarbonatesolution was added, followed by extraction with ethyl acetate. Theorganic layer was washed with water and saturated brine and dried overanhydrous sodium sulfate and the solvent was concentrated under reducedpressure. The residue obtained was purified by silica gel chromatography[chloroform:methanol=50:1 (v/v)) to give the title compound (309 mg,89%) as a colorless solid.

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.43-0.48 (2H, m), 0.56-0.62 (2H,m), 0.93 (6H, d, J=7.1 Hz), 1.77 (3H, s), 2.03-2.18 (1H, m), 3.30 (3H,s), 2.60-2.67 (2H, m), 2.73-2.78 (2H, m), 3.34 (2H, brs), 3.54 (2H,brs), 3.68-3.78 (1H, m), 3.80-3.95 (1H, m), 5.04 (1H, t, J=7.8 Hz), 5.34(1H, d, J=53.0 Hz), 5.50 (1H, s), 6.75 (1H, d, J=8.0 Hz), 6.93 (1H, d,J=9.0 Hz), 7.16 (1H, d, J=8.3 Hz), 7.33 (1H, t, J=8.0 Hz), 7.64 (1H, dd,J=8.3, 2.3 Hz), 8.25 (1H, d, J=2.3 Hz).

MS (ESI) m/z: 689 [(M+1)]⁺.

The compound obtained in Step 2 of Example 2 or the compound obtained inStep 2 of Example 3 was reacted in the same way as in Example 15 aboveto give the compounds in the following table.

TABLE 14 Example 16

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.20-0.25 (1H, m), 0.46-0.51 (1H,m), 0.61-0.65 (1H, m), 0.84-0.90 (1H, m), 0.91 (3H, d, J = 7.1 Hz), 0.94(3H, d, J = 7.1 Hz), 1.25-1.28 (3H, m), 1.77 (3H, s), 2.08 (3H, s),2.10-2.21 (1H, m), 2.50-2.64 (4H, m), 3.18-3.26 (1H, m), 3.53-3.60 (1H,m), 3.68-3.78 (1H, m), 3.80-3.95 (1H, m), 4.37-4.40 (1H, m), 4.98 (1H,t, J = 7.8 Hz), 5.34 (1H, d, J = 53 Hz), 5.49 (1H, s), 6.75 (1H, d, J =8.0 Hz), 6.93 (1H, d, J = 9.0 Hz), 7.16 (1H, d, J = 8.3 Hz), 7.33 (1H,t, J = 8.0 Hz), 7.64 (1H, dd, J = 8.3, 2.4 Hz), 8.25 (1H, d, J = 2.4Hz). MS (ESI) m/z: 703 [(M + 1)]⁺. Example 17

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.30 (1H, br), 0.45-0.50 (1H, m),0.60-0.64 (1H, m), 0.85-0.90 (1H, m), 0.92 (3H, d, J = 7.1 Hz), 0.93(3H, d, J = 7.1 Hz), 1.25-1.35 (3H, m), 1.77 (3H, m), 1.92-2.07 (1H, m),2.09 (3H, s), 2.62-2.68 (4H, m), 3.22-3.34 (1H, m), 3.43-3.51 (1H, m),3.67-3.79 (1H, m), 3.86-3.95 (1H, m), 4.27-4.35 (1H, m), 5.00 (1H, t, J= 7.8 Hz), 5.34 (1H, d, J = 53 Hz), 5.49 (1H, s), 6.74 (1H, d, J = 8.3Hz), 6.92 (1H, d, J = 9.5 Hz), 7.16 (1H, d, J = 8.3 Hz), 7.33 (1H, t, J= 8.0 Hz), 7.64 (1H, dd, J = 8.3, 2.5 Hz), 8.25 (1H, d, J = 2.5 Hz). MS(ESI) m/z: 703 [(M + 1)]⁺.

The following compounds were synthesized according to the descriptionsof the general production methods and Examples 1 to 12.

TABLE 15 Example 18

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.43-0.49 (4H, m), 0.88 (3H, d, J= 7.1 Hz), 0.91 (3H, d, J = 7.1 Hz), 1.73 (3H, s), 2.01-2.31 (1H, m),2.55-2.66 (2H, m), 2.71-2.76 (2H, m), 3.31-3.36 (2H, m), 3.43-3.47 (2H,m), 3.67-3.80 (1H, m), 3.85-3.93 (1H, m), 5.03 (1H, t, J = 7.8 Hz), 5.32(1H, d, J = 53 Hz), 5.39 (1H, s), 6.89 (2H, d, J = 8.5 Hz), 7.03-7.07(2H, m), 7.11 (2H, d, J = 8.5 Hz), 7.22-7.25 (2H, m). MS (ESI) m/z: 656[(M + 1)]⁺. Example 19

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.35-0.39 (2H, m), 0.54-0.59 (2H,m), 0.86 (3H, d, J = 7.1 Hz), 0.92 (3H, d, J = 7.1 Hz), 1.20 (3H, brd, J= 6.1 Hz), 1.73 (3H, s), 2.05-2.21 (1H, m), 2.51-2.65 (3H, m), 2.88-2.91(1H, m), 3.23 (1H, br), 3.45 (1H, br), 3.67-3.71 (1H, m), 3.76-3.93 (1H,m), 4.31 (1H, br), 4.96 (1H, br), 5.34 (1H, d, J = 53 Hz), 5.38 (1H, s),6.89 (2H, d, J = 7.5 Hz), 7.04-7.07 (2H, m), 7.11 (2H, d, J = 7.5 Hz),7.22-7.25 (2H, m). MS (ESI) m/z: 670 [(M + 1)]⁺. Example 20

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.35 (1H, br), 0.56 (2H, br), 0.86(1H, br), 0.88 (3H, d, J = 7.1 Hz), 0.90 (3H, d, J = 7.1 Hz), 1.25 (3H,brs), 1.73 (3H, s), 2.01 (1H, br), 2.57-2.69 (3H, m), 2.87-2.92 (1H, m),3.35 (2H, br), 3.66-3.78 (1H, m), 3.85-3.93 (1H, m), 4.52 (1H, br), 5.00(1H, t, J = 8.1 Hz), 5.32 (1H, d, J = 53 Hz), 5.38 (1H, s), 6.88 (2H, d,J = 8.6 Hz), 7.03-7.07 (2H, m), 7.10 (2H, d, J = 8.6 Hz), 7.22-7.25 (2H,m). MS (ESI) m/z: 670 [(M + 1)]⁺.

TABLE 16 Example 21

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.33-0.38 (2H, m), 0.52-0.58 (2H,m), 0.80-0.83 (3H, m), 0.86 (3H, d, J = 7.1 Hz), 0.92 (3H, d, J = 7.1Hz), 1.66-1.70 (2H, m), 1.73 (3H, s), 2.10-2.24 (1H, m), 2.43-2.65 (3H,m), 2.73-2.78 (1H, m), 3.65 (2H, br), 3.70-3.80 (1H, m), 3.82-3.94 (1H,m), 4.05 (1H, br), 4.98 (1H, br), 5.36 (1H, d, J = 53 Hz), 5.38 (1H, s),6.90 (2H, d, J = 8.3 Hz), 7.04-7.07 (2H, m), 7.11 (2H, d, J = 8.3 Hz),7.22-7.25 (2H, m). MS (ESI) m/z: 684 [(M + 1)]⁺. Example 22

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.32-0.36 (1H, m), 0.50-0.57 (3H,m), 0.83-0.86 (3H, m), 0.88 (3H, d, J = 7.1 Hz), 0.91 (3H, d, J = 7.1Hz), 1.62-1.68 (2H, m), 1.73 (3H, s), 2.09-2.22 (1H, m), 2.59-2.64 (2H,m), 2.81-2.85 (2H, m), 3.10 (1H, br), 3.50 (1H, br), 3.66-3.78 (1H, m),3.85-3.93 (1H, m), 4.21 (1H, br), 5.02 (1H, t, J = 8.0 Hz), 5.36 (1H, d,J = 53 Hz), 5.38 (1H, s), 6.89 (2H, d, J = 8.3 Hz), 7.03-7.07 (2H, m),7.10 (2H, d, J = 8.3 Hz), 7.22-7.25 (2H, m). MS (ESI) m/z: 684 [(M +1)]⁺. Example 23

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.44-0.48 (4H, m), 0.91 (3H, d, J= 7.1 Hz), 0.93 (3H, d, J = 7.1 Hz), 1.74 (3H, s), 2.02-2.34 (1H, m),2.53-2.66 (2H, m), 2.72-2.76 (2H, m), 3.32-3.37 (2H, m), 3.42-3.48 (2H,m), 3.67-3.80 (1H, m), 3.86-3.93 (1H, m), 5.03 (1H, t, J = 7.8 Hz), 5.33(1H, d, J = 53 Hz), 5.43 (1H, s), 6.71 (1H, d, J = 8.0 Hz), 6.88 (1H, d,J = 10.0 Hz), 7.07-7.10 (2H, m), 7.24-7.29 (3H, m). MS (ESI) m/z: 674[(M + 1)]⁺.

TABLE 17 Example 24

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.33-0.38 (2H, m), 0.54-0.58 (2H,m), 0.89 (3H, d, J = 6.8 Hz), 0.93 (3H, d, J = 6.8 Hz), 1.19 (3H, brd, J= 6.5 Hz), 1.73 (3H, s), 2.07-2.19 (1H, m), 2.54-2.64 (3H, m), 2.87-2.91(1H, m), 3.19 (1H, br), 3.44 (1H, br), 3.67-3.70 (1H, m), 3.76-3.93 (1H,m), 4.31 (1H, br), 4.95 (1H, br), 5.35 (1H, d, J = 53.6 Hz), 5.44 (1H,s), 6.71 (1H, d, J = 7.8 Hz), 6.88 (1H, d, J = 9.1 Hz), 7.07-7.10 (2H,m), 7.25-7.30 (3H, m). MS (ESI) m/z: 688 [(M + 1)]⁺. Example 25

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.35 (1H, br), 0.55 (2H, br), 0.88(1H, br), 0.91 (3H, d, J = 6.9 Hz), 0.92 (3H, d, J = 6.9 Hz), 1.20 (3H,brs), 1.73 (3H, s), 1.98 (1H, br), 2.59-2.68 (3H, m), 2.87-2.91 (1H, m),3.41 (2H, br), 3.68-3.78 (1H, m), 3.85-3.93 (1H, m), 4.50 (1H, br), 5.00(1H, t, J = 8.2 Hz), 5.33 (1H, d, J = 53 Hz), 5.38 (1H, s), 6.71 (1H, d,J = 8.2 Hz), 6.88 (1H, d, J = 10.0 Hz), 7.08-7.11 (2H, m), 7.24-7.29(3H, m). MS (ESI) m/z: 688 [(M + 1)]⁺. Example 26

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.44-0.48 (4H, m), 0.90 (3H, d, J= 7.1 Hz), 0.91 (3H, d, J = 7.1 Hz), 1.76 (3H, s), 2.01-2.36 (1H, m),2.53-2.66 (2H, m), 2.72-2.76 (2H, m), 3.28-3.37 (2H, m), 3.44-3.48 (2H,m), 3.67-3.79 (1H, m), 3.86-3.94 (1H, m), 5.04 (1H, t, J = 7.8 Hz), 5.33(1H, d, J = 53 Hz), 5.46 (1H, s), 6.92 (2H, d, J = 8.3 Hz), 7.11-7.18(3H, m), 7.62 (1H, dd, J = 8.3, 2.4 Hz), 8.23 (1H, d, J = 2.4 Hz). MS(ESI) m/z: 657 [(M + 1)]⁺.

TABLE 18 Example 27

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.36-0.38 (2H, m), 0.54-0.58 (2H,m), 0.89 (3H, d, J = 7.1 Hz), 0.92 (3H, d, J = 7.1 Hz), 1.20 (3H, brd, J= 5.8 Hz), 1.76 (3H, s), 2.05-2.21 (1H, m), 2.50-2.65 (3H, m), 2.88-2.91(1H, m), 3.24 (1H, br), 3.44 (1H, br), 3.67-3.71 (1H, m), 3.76-3.94 (1H,m), 4.31 (1H, br), 4.97 (1H, br), 5.34 (1H, d, J = 53 Hz), 5.45 (1H, s),6.92 (2H, d, J = 8.3 Hz), 7.11-7.17 (3H, m), 7.62 (1H, dd, J = 8.3, 2.7Hz), 8.22 (1H, d, J = 2.7 Hz). MS (ESI) m/z: 671 [(M + 1)]⁺. Example 28

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.36 (1H, br), 0.56 (2H, br), 0.85(1H, br), 0.90 (6H, d, J = 7.1 Hz), 1.25 (3H, brs), 1.76 (3H, s), 2.17(1H, br), 2.60-2.69 (3H, m), 2.88-2.92 (1H, m), 3.39 (2H, br), 3.66-3.78(1H, m), 3.86-3.94 (1H, m), 4.45 (1H, br), 5.00 (1H, t, J = 8.3 Hz),5.33 (1H, d, J = 54 Hz), 5.45 (1H, s), 6.91 (2H, d, J = 8.4 Hz),7.11-7.17 (3H, m), 7.61 (1H, dd, J = 8.4, 2.7 Hz), 8.23 (1H, d, J = 2.7Hz). MS (ESI) m/z: 671 [(M + 1)]⁺. Example 29

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.34-0.38 (2H, m), 0.52-0.57 (2H,m), 0.81-0.83 (3H, m), 0.88 (3H, d, J = 7.1 Hz), 0.91 (3H, d, J = 7.1Hz), 1.68-1.72 (2H, m), 1.76 (3H, s), 2.12-2.27 (1H, m), 2.50-2.65 (3H,m), 2.73-2.76 (1H, m), 3.69 (2H, br), 3.70-3.80 (1H, m), 3.82-3.95 (1H,m), 4.12 (1H, br), 4.97 (1H, br), 5.36 (1H, d, J = 54 Hz), 5.45 (1H, s),6.92 (2H, d, J = 8.3 Hz), 7.11-7.18 (3H, m), 7.62 (1H, dd, J = 8.3, 2.2Hz), 8.23 (1H, d, J = 2.2 Hz). MS (ESI) m/z: 685 [(M + 1)]⁺.

TABLE 19 Example 30

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.33-0.37 (1H, m), 0.51-0.57 (3H,m), 0.82-0.87 (3H, m), 0.90 (6H, d, J = 7.1 Hz), 1.68-1.72 (2H, m), 1.76(3H, s), 1.99-2.13 (1H, m), 2.60-2.65 (2H, m), 2.81-2.94 (2H, m), 3.11(1H, br), 3.49 (1H, br), 3.66-3.78 (1H, m), 3.86-3.95 (1H, m), 4.21 (1H,br), 5.02 (1H, t, J = 8.0 Hz), 5.36 (1H, d, J = 55 Hz), 5.45 (1H, s),6.92 (2H, d, J = 8.0 Hz), 7.11-7.17 (3H, m), 7.61 (1H, dd, J = 8.0, 2.4Hz), 8.22 (1H, d, J = 2.4 Hz). MS (ESI) m/z: 685 [(M + 1)]⁺. Example 31

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.55 (4H, brs), 0.92 (3H, d, J =7.2 Hz), 0.94 (3H, d, J = 7.2 Hz), 1.77 (3H, s), 2.10-2.30 (2H, m),2.53-2.66 (2H, m), 2.86-2.94 (1H, m), 3.05-3.20 (1H, m), 3.39 (1H, br),3.51-3.54 (1H, m), 3.76-3.98 (3H, m), 4.97 (1H, br), 5.04 (1H, t, J =7.8 Hz), 5.37 (1H, d, J = 54 Hz), 5.48 (1H, s), 6.75 (1H, d, J = 8.3Hz), 6.92 (1H, d, J = 8.8 Hz), 7.14 (1H, d, J = 8.3 Hz), 7.31 (1H, t, J= 8.0 Hz), 7.63 (1H, dd, J = 8.3, 2.5 Hz), 8.24 (1H, d, J = 2.5 Hz). MS(ESI) m/z: 707 [(M + 1)]⁺.

Reference Example 1

Step 1: ethyl 2-(6-chloropyridin-3-yl)propanoate

A methanol (200 ml) solution of ammonium chloride (6.88 g, 129 mmol) and1-(6-chloropyridin-3-yl)ethanone (10.0 g, 64.3 mmol) was added to anaqueous concentrated ammonia solution (100 ml) of potassium cyanide(10.7 g, 161 mmol) under ice cooling and the resulting mixture wasreturned to room temperature while being stirred for 3 days. Thereaction solution was concentrated under reduced pressure and theresidue was subjected to extraction with dichloromethane. The organiclayer was dried over anhydrous magnesium sulfate and then the solventwas evaporated under reduced pressure. Concentrated hydrochloric acid(100 ml) was added to the residue obtained under ice cooling and thenthe resulting mixture was heated to reflux for 2 hours. The reactionmixture was concentrated under reduced pressure and then subjected toazeotropic distillation with toluene and thionyl chloride (10 ml) wasadded dropwise to an ethanol (100 ml) solution of the residue obtainedunder ice cooling. The resulting mixture was heated to reflux for 3hours, then the reaction mixture was concentrated under reduced pressureand the residue obtained was diluted with dichloromethane and thenwashed with saturated aqueous sodium bicarbonate solution and saturatedbrine. The organic layer was dried over anhydrous magnesium sulfate andthe solvent was evaporated under reduced pressure. The residue obtainedwas purified by silica gel column chromatography (ethyl acetate) to givethe title compound (7.24 g, 49%) as a pale yellow oil.

¹H-NMR (400 MHz, CDCl₃) δ: 1.25 (3H, t, J=7.1 Hz), 1.70 (3H, s), 1.98(2H, brs), 4.19 (2H, q, J=7.1 Hz), 7.30 (1H, d, J=8.3 Hz), 7.85 (1H, dd,J=8.3, 2.4 Hz), 8.56 (1H, d, J=2.4 Hz).

Step 2: ethyl2-[(tert-butoxycarbonyl)amino]-2-(6-chloropyridin-3-yl)propanoate

Triethylamine (1.22 ml, 8.75 mmol) and di-tert-butyl dicarbonate (1.12ml, 4.81 mmol) were added to a tetrahydrofuran (20 ml) solution of thecompound (1.00 g, 4.37 mmol) obtained in Step 1 above and the resultingmixture was heated to reflux for 18 hours. Further di-tert-butyldicarbonate (0.51 ml, 2.19 mmol) was added and the resulting mixture washeated to reflux for 18 hours. The reaction mixture was concentratedunder reduced pressure and then the residue obtained was diluted withethyl acetate, washed with 10% aqueous citric acid solution andsaturated brine, and then dried over anhydrous magnesium sulfate. Thesolvent was evaporated under reduced pressure and the residue obtainedwas purified by silica gel column chromatography [n-hexane:ethylacetate=2:18 (v/v)] to give the title compound (1.20 g, 84%) as a paleyellow oil.

¹H-NMR (400 MHz, CDCl₃) δ: 1.19 (3H, t, J=7.2 Hz), 1.38 (9H, brs), 1.99(3H, s), 4.08-4.26 (2H, m), 7.30 (1H, d, J=8.5 Hz), 7.74 (1H, dd, J=8.5,2.7 Hz), 8.47 (1H, d, J=2.7 Hz)

Step 3: tert-butyl[1-(6-chloropyridin-3-yl)-1-methyl-2-oxoethyl]carbamate

A tetrahydrofuran (100 ml) solution of the compound (5.70 g, 17.3 mmol)obtained in Step 2 above was added dropwise to a tetrahydrofuran (20 ml)suspension of lithium aluminum hydride (1.43 g, 34.7 mmol) under icecooling and the resulting mixture was stirred at the same temperaturefor 1 hour. 1 N aqueous sodium hydroxide solution (6 ml) was added tothe reaction mixture under ice cooling and the precipitated insolublematter was removed by filtration. The filtrate was concentrated underreduced pressure and the residue obtained was dissolved in dimethylsulfoxide (100 ml). Triethylamine (60 ml) and a sulfur trioxide-pyridinecomplex (5.52 g, 34.7 mmol) were added at room temperature and theresulting mixture was stirred for 2 hours. The reaction mixture waspoured into water, followed by extraction with ethyl acetate. Theorganic layer was washed with saturated brine. The organic layer wasdried over anhydrous magnesium sulfate, then the solvent was evaporatedunder reduced pressure and the residue obtained was purified by silicagel column chromatography [n-hexane:ethyl acetate=2:1 (v/v)] to give thetitle compound (2.77 g, 56%) as a pale yellow oil.

¹H-NMR (400 MHz, CDCl₃) δ: 1.43 (9H, s), 1.79 (3H, s), 5.73 (1H, brs),7.36 (1H, d, J=8.5 Hz), 7.69 (1H, dd, J=8.5, 2.7 Hz), 8.42 (1H, s), 9.34(1H, s).

Step 4: tert-butyl[2-(4-chloro-3-fluorophenyl)-1-(6-chloropyridin-3-yl)-2-hydroxy-1-methylethyl]carbamate

A tetrahydrofuran (20 ml) solution of the compound (6.47 g, 22.7 mmol)obtained in Step 3 above was added dropwise to 4-chloro-3-fluorophenylmagnesium bromide (0.5 M tetrahydrofuran solution) (100 ml, 50.0 mmol)under ice cooling and the resulting mixture was stirred at the sametemperature for 1 hour. An aqueous solution of saturated ammoniumchloride was added to the reaction mixture to terminate the reaction,followed by extraction with ethyl acetate. The organic layer was driedover anhydrous magnesium sulfate and then the solvent was evaporatedunder reduced pressure. An n-hexane/ethyl acetate mixed solvent wasadded to the residue obtained and the resulting precipitate wascollected by filtration and dried to give the title compound (7.92 g,84%) as a white solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.45 (9H, brs), 1.56 (3H, brs), 5.08 (2H,brs), 6.86 (1H, brs), 7.09 (1H, d, J=8.8 Hz), 7.31-7.35 (2H, m), 7.68(1H, d, J=7.3 Hz), 8.44 (1H, s).

MS (API) m/z: 415 [(M+1)⁺].

Step 5: tert-butyl[2-(4-chloro-3-fluorophenyl)-1-(6-chloropyridin-3-yl)-1-methyl-2-oxoethyl]carbamate

Acetic anhydride (2.3 ml, 24.7 mmol) was added to a dimethyl sulfoxide(8 ml) solution of the compound (1.14 g, 2.75 mmol) obtained in Step 4above under ice cooling and the resulting mixture was gradually returnedto room temperature while being stirred for 18 hours. The reactionmixture was diluted with ethyl acetate, washed with 6% aqueous sodiumperchlorate solution, 10% aqueous sodium thiosulfate solution, andsaturated brine, and then dried over anhydrous magnesium sulfate. Thesolvent was evaporated under reduced pressure and the residue obtainedwas purified by silica gel column chromatography [n-hexane:ethylacetate=4:1 (v/v)] to give the title compound (1.10 g, 97%) as a paleyellow solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.31 (9H, s), 2.03 (3H, s), 6.28 (1H, brs),7.26-7.37 (3H, m), 7.49 (1H, d, J=9.5 Hz), 7.71 (1H, d, J=6.3 Hz), 8.45(1H, s).

Step 6:2-amino-1-(4-chloro-3-fluorophenyl)-2-(6-chloropyridin-3-yl)propan-1-one

A dioxane (1 ml) solution of the compound (1.23 g, 2.55 mmol) obtainedin Step 5 above was added to a 4 N hydrochloric acid/dioxane solution (5ml) under ice cooling and the resulting mixture was gradually warmed andstirred at room temperature for 2 hours. The reaction mixture wasdiluted with water and washed with an n-hexane/ethyl acetate mixedsolvent and then the aqueous layer was made alkaline by the addition of15% aqueous sodium hydroxide solution, followed by extraction withdichloromethane. The organic layer was dried over anhydrous magnesiumsulfate, then the solvent was evaporated under reduced pressure and theresidue obtained was purified by silica gel column chromatography[n-hexane:ethyl acetate=1:1 (v/v)] to give the title compound (0.75 g,95%) as a pale yellow solid.

MS (API) m/z: 313 [(M+1)⁺].

Step 7:2-chloro-5-[4-(4-chloro-3-fluorophenyl)-3-methyl-1,1-dioxido-2,3-dihydro-1,2,5-thiadiazol-3-yl]pyridine

A molecular sieve 4A (4.5 g), sulfamide (2.75 g, 28.6 mmol), and1,8-diazabicyclo[5.4.0]undec-7-ene (1.45 g, 9.52 mmol) were added to adioxane (60 ml) solution of the compound (2.99 g, 9.55 mmol) obtained inStep 6 above and the resulting mixture was stirred under heating at 95°C. for 18 hours. Further sulfamide (2.75 g, 28.6 mmol) was added and theresulting mixture was stirred under heating for 24 hours. The reactionmixture was concentrated under reduced pressure and then the residueobtained was diluted with ethyl acetate, washed with 10% aqueous citricacid solution and saturated brine, and then dried over anhydrousmagnesium sulfate. A diisopropyl ether/ethyl acetate mixed solvent wasadded to the residue obtained and the resulting precipitate wascollected by filtration and dried to give the title compound (2.86 g,80%) as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 2.25 (3H, s), 7.29-7.47 (3H, m), 7.59 (1H,dd, J=9.5, 2.0 Hz), 7.68 (1H, dd, J=8.5, 2.9 Hz), 8.53 (1H, d, J=2.9Hz).

Step 8:2-chloro-5-[(3R*,4S*)-4-(4-chloro-3-fluorophenyl)-3-methyl-1,1-dioxido-1,2,5-thiadiazolidin-3-yl]pyridine

Sodium borohydride (1.00 g, 26.4 mmol) was gradually added to an ethanol(63 ml) solution of the compound (7.13 g, 19.1 mmol) obtained in Step 7above under ice cooling and the resulting mixture was stirred at thesame temperature for 2 hours. The reaction mixture was concentratedunder reduced pressure and then the residue obtained was diluted withethyl acetate, washed with 1 N aqueous hydrochloric acid solution andsaturated brine, and then dried over anhydrous magnesium sulfate. Thesolvent was evaporated under reduced pressure and the residue obtainedwas purified by silica gel column chromatography [n-hexane:ethylacetate=1:2 (v/v)] to give the title compound (4.36 g, 59%) as a paleyellow solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.85 (3H, s), 3.23-3.32 (4H, m), 4.92 (1H, d,J=5.6 Hz), 6.74-6.76 (1H, m), 6.80-6.83 (1H, m), 7.23-7.26 (2H, m),7.55-7.60 (1H, m), 7.94-7.95 (1H, m).

Step 9:(1R*,2S*)-1-(4-chloro-3-fluorophenyl)-2-(6-chloropyridin-3-yl)propane-1,2-diamine

Ethylenediamine (7.40 ml, 111 mmol) was added to a dioxane (80 ml)solution of the compound (4.15 g, 11.0 mmol) obtained in Step 8 aboveand the resulting mixture was stirred under heating at 100° C. for 18hours. The reaction mixture was concentrated under reduced pressure andthe residue obtained was diluted with chloroform, washed with 1 Naqueous sodium hydroxide solution and saturated brine, and then driedover anhydrous magnesium sulfate. The solvent was evaporated underreduced pressure and the residue obtained was purified by silica gelcolumn chromatography [chloroform:methanol=93:7 (v/v)] to give the titlecompound (3.35 g, 97%) as a pale yellow oil.

¹H-NMR (400 MHz, CDCl₃) δ: 1.54 (3H, s), 1.58 (4H, brs), 4.08 (1H, s),6.72 (1H, dd, J=8.3, 2.0 Hz), 6.96 (1H, dd, J=10.3, 2.0 Hz), 7.21 (1H,t, J=7.9 Hz), 7.23 (1H, dd, J=8.3, 0.7 Hz), 7.59 (1H, dd, J=8.5, 2.7Hz), 8.36 (1H, dd, J=2.7, 0.7 Hz).

MS (ESI) m/z: 314 [(M+1)⁺].

Step 10:(1R,2S)-1-(4-chloro-3-fluorophenyl)-2-(6-chloropyridin-3-yl)propane-1,2-diamine,

L-(+)-tartaric acid (6.3 g, 42.0 mmol) was added to an ethanol (200 ml)solution of the compound (12.6 g, 40.0 mmol) obtained in Step 9 aboveand the resulting mixture was heated to reflux at 110° C. for 30minutes. Water (8 ml) was added, the resulting mixture was furtherheated to reflux for 10 minutes, then returned to room temperature, andleft overnight and then the precipitated solid was collected byfiltration. The solid obtained was made into an alkaline solution by theaddition of 5 N aqueous sodium hydroxide solution, followed byextraction with diethyl ether. The organic layer was dried overpotassium carbonate and then the solvent was evaporated under reducedpressure to give the title compound (5.68 g, 45%) as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.54 (3H, s), 1.56 (4H, brs), 4.08 (1H, s),6.72 (1H, dd, J=8.3, 2.0 Hz), 6.96 (1H, dd, J=10.4, 2.1 Hz), 7.20-7.24(2H, m), 7.59 (1H, dd, J=8.4, 2.6 Hz), 8.36 (1H, dd, J=2.7, 0.7 Hz).

MS (ESI) m/z: 314 [(M+1)⁺].

[α]_(D)=+55.5° (C=1.00, chloroform, 20° C.).

Step 11:(4S,5R)-5-(4-chloro-3-fluorophenyl)-4-(6-chloropyridin-3-yl)-4-methylimidazolidine-2-thione

Carbon disulfide (0.58 ml, 9.21 mmol) was added to an ethanol (30 ml)solution of the compound (1.93 g, 6.14 mmol) obtained in Step 10 aboveat room temperature and the resulting mixture was heated to reflux for20 hours. After cooling, the reaction mixture was concentrated underreduced pressure and the residue obtained was diluted with ethylacetate, washed with saturated brine, and then dried over anhydrousmagnesium sulfate. The solvent was evaporated under reduced pressure andthe residue obtained was purified by silica gel column chromatography[chloroform:methanol=40:1 (v/v)] to give the title compound (1.94 g,89%) as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.94 (3H, s), 5.00 (1H, s), 6.67 (1H, d,J=7.8 Hz), 6.68 (1H, brs), 6.75 (1H, dd, J=9.5, 2.0 Hz), 7.10 (1H, brs),7.15 (1H, d, J=8.3 Hz), 7.21 (1H, t, J=7.8 Hz), 7.31 (1H, dd, J=8.5, 2.7Hz), 8.01 (1H, d, J=2.2 Hz).

MS (ESI) m/z: 356 [(M+1)⁺].

Step 12: ethyl(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole-2-carboxylate

Ethyl 2-chloro-4-methyl-3-oxopentanoate (1.36 g, 7.09 mmol) was added toan ethanol (20 ml) solution of the compound (1.94 g, 5.45 mmol) obtainedin Step 11 above at room temperature and the resulting mixture washeated to reflux for 16 hours. The reaction mixture was returned to roomtemperature and the reaction solvent was evaporated under reducedpressure. The residue obtained was diluted with ethyl acetate and theorganic layer was washed with saturated aqueous sodium bicarbonatesolution and saturated brine and dried over anhydrous magnesium sulfate.The solvent was evaporated under reduced pressure and the residueobtained was purified by silica gel column chromatography[n-hexane:ethyl acetate=3:1 (v/v)] to give the title compound (2.06 g,76%) as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.90 (3H, d, J=7.3 Hz), 1.04 (3H, d, J=7.1Hz), 1.34 (3H, t, J=7.2 Hz), 1.83 (3H, s), 4.26 (2H, q, J=7.2 Hz), 5.10(1H, s), 6.51-6.62 (2H, m), 7.04 (1H, d, J=8.3 Hz), 7.16 (1H, t, J=7.7Hz), 7.51-7.55 (1H, m), 8.20-8.22 (1H, m).

MS (ESI) m/z: 494 [(M+1)⁺].

Step 13:(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole-2-carboxylicacid

1 N aqueous sodium hydroxide solution (6 ml) was added to an ethanol (30ml) solution of the compound (2.06 g, 4.17 mmol) obtained in Step 12above and the resulting mixture was stirred under heating at 60° C. for16 hours. After cooling, the solvent was concentrated under reducedpressure and the residue obtained was diluted with water and then washedwith diethyl ether. The aqueous layer was made into an acidic solutionby the gradual addition of 1 N aqueous hydrochloric acid solution underice cooling, followed by extraction with ethyl acetate. The organiclayer was washed with saturated brine and then dried over anhydrousmagnesium sulfate. The solvent was evaporated under reduced pressure, adiethyl ether/n-hexane mixed solvent was added to the residue and theresulting precipitate was collected by filtration and dried to give thetitle compound (1.51 g, 87%) as a pale yellow solid.

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.79 (3H, d, J=7.1 Hz), 0.98 (3H, d, J=7.1Hz), 1.73 (3H, s), 3.32-3.40 (1H, m), 5.71 (1H, s), 6.38-6.48 (1H, m),7.23 (1H, d, J=8.3 Hz), 7.34-7.46 (2H, m), 7.66 (1H, dd, J=8.3, 2.4 Hz),8.25 (1H, d, J=2.4 Hz).

MS (ESI) m/z: 466 [(M+1)⁺].

[α]_(D)=+120.5° (C=1.00, methanol, 25° C.).

Reference Example 2

Step 1: ethylN-benzyl-N-[(1-{[(benzyloxy)carbonyl]amino}cyclopropyl)carbonyl]glycinate

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21.0 g, 110mmol) and 1-hydroxybenzotriazole (2.70 g, 20 mmol) were added to adichloromethane (235 ml) solution of1-{[(benzyloxy)carbonyl]amino}cyclopropanecarboxylic acid (23.5 g, 100mmol) and ethyl N-benzylglycinate (19.3 g, 100 mmol) under ice coolingand the resulting mixture was stirred at room temperature for 24 hours.The solvent was concentrated under reduced pressure and then the residuewas diluted with ethyl acetate, washed with 1 N aqueous hydrochloricacid solution, saturated aqueous sodium bicarbonate solution, andsaturated brine, and then dried over anhydrous sodium sulfate. Thesolvent was evaporated under reduced pressure and the residue obtainedwas purified by silica gel column chromatography [n-hexane:ethylacetate=2:1 (v/v)] to give the title compound (35.7 g, 87%) as acolorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.13 (2H, br), 1.22 (3H, t, J=7.4 Hz), 1.25(2H, br), 1.66 (2H, s), 3.91 (1H, br), 4.12 (2H, q, J=7.4 Hz), 4.91 (2H,brs), 5.36 (2H, brs), 7.19-7.31 (10H, m).

Step 2: 7-benzyl-4,7-diazaspiro[2.5]octane-5,8-dione

5% palladium carbon (3.6 g) was added to an ethanol (700 ml) solution ofthe compound (35.5 g, 86.5 mmol) obtained in Step 1 above and theresulting mixture was subjected to catalytic reduction for 2 hours in ahydrogen atmosphere. The catalyst was removed by filtration throughcelite, then the filtrate was concentrated under reduced pressure andthe residue obtained was purified by silica gel column chromatography[ethyl acetate:n-hexane=1:1 (v/v)] to give the title compound (20 g,100%) as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.93-1.00 (2H, m), 1.55-1.59 (2H, m), 3.91(2H, s), 4.60 (2H, s), 7.25-7.37 (5H, m), 7.86 (1H, brs).

MS (ESI) m/z: 231 [(M+H)⁺].

Step 3: 7-benzyl-4,7-diazaspiro[2.5]octane

A borane-tetrahydrofuran complex (0.93 M tetrahydrofuran solution) (375ml, 0.35 mol) was added to a tetrahydrofuran (200 ml) solution of thecompound (20 g, 86.8 mmol) obtained in Step 2 above under ice coolingand then the resulting mixture was heated to reflux for 19 hours.Methanol (130 ml) was added to the reaction mixture under ice cooling,the resulting mixture was stirred for 60 minutes and then the solventwas concentrated under reduced pressure. Ethanol (450 ml), water (150ml), and triethylamine (150 ml) were added to the residue obtained, theresulting mixture was heated to reflux for 2 hours and then the solventwas concentrated under reduced pressure. The residue obtained wasdiluted with ethyl acetate, washed with saturated aqueous sodiumbicarbonate solution and saturated brine, and then dried over anhydroussodium sulfate. The solvent was evaporated under reduced pressure andthe residue obtained was purified by silica gel column chromatography[chloroform:methanol=10:1 (v/v)] to give the title compound (10.4 g,59%) as a colorless oil.

¹H-NMR (400 MHz, CDCl₃) δ: 0.41-0.44 (2H, m), 0.57-0.60 (2H, m), 1.49(1H, br), 2.22 (2H, s), 2.45 (2H, brs), 2.97 (2H, t, J=4.9 Hz), 3.50(2H, s), 7.22-7.32 (5H, m).

Step 4: 7-benzyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

Trifluoroacetic anhydride (8.50 ml, 61.1 mmol) was added dropwise to adichloromethane (200 ml) solution of the compound (10.3 g, 50.9 mmol)obtained in Step 3 above and triethylamine (17 ml, 122 mmol) under icecooling and the resulting mixture was stirred at the same temperaturefor 1 hour. Saturated aqueous sodium bicarbonate solution was added tothe reaction mixture and the resulting mixture was diluted withchloroform, then washed with saturated brine, and dried over anhydroussodium sulfate. The solvent was evaporated under reduced pressure togive the title compound (15.5 g, 100%) as a colorless oil.

MS (ESI) m/z: 299 [(M+H)⁺].

Step 5: 4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane hydrochloride

1 N hydrochloric acid/ethanol (105 ml, 105 mmol) and 5% palladium carbon(3 g) were added to an ethanol (250 ml) solution of the compound (15.5g, 51 mmol) obtained in Step 4 above and the resulting mixture wassubjected to catalytic reduction for 15 hours in a hydrogen atmosphere.The catalyst was removed by filtration through celite and then thefiltrate was concentrated under reduced pressure. An ethanol/diethylether mixed solvent was added to the residue obtained and the depositedsolid was collected by filtration to give the title compound (10.3 g,83%) as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 80° C.) δ: 1.18 (4H, s), 3.16 (2H, s), 3.25(2H, t, J=5.1 Hz), 3.89 (2H, brs), 9.71 (2H, br).

MS (ESI) m/z: 209 [(M+H)⁺].

Reference Example 3

Step 1: tert-butyl(2S,4R)-4-fluoro-2-{[4-(trifluoroacetyl)-4,7-diazaspiro[2.5]oct-7-yl]carbonyl}pyrrolidine-1-carboxylate

1-hydroxybenzotriazole (46 mg, 0.34 mmol) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (502 mg,2.61 mmol) were added to a dimethylformamide (8 ml) solution of(4R)-1-(tert-butoxycarbonyl)-4-fluoro-L-proline (400 mg, 1.71 mmol) andthe resulting mixture was stirred at room temperature for 15 minutes.Subsequently, the compound (460 mg, 2.05 mmol) obtained in Step 5 ofReference Example 2 and diisopropylethylamine (0.45 ml, 2.57 mmol) wereadded and the resulting mixture was stirred at room temperature for 16hours. The reaction mixture was diluted with ethyl acetate and theorganic layer was washed with saturated aqueous sodium bicarbonatesolution and saturated brine and then dried over anhydrous sodiumsulfate. The solvent was evaporated under reduced pressure and theresidue obtained was purified by silica gel column chromatography[n-hexane:ethyl acetate=1:1 (v/v)] to give the title compound (560 mg,77%) as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.98-1.10 (4H, m), 1.37 (9H, s),1.95-2.09 (1H, m), 2.49-2.54 (1H, m), 3.41-3.54 (3H, m), 3.60-3.75 (5H,m), 4.73-4.80 (1H, m), 5.26 (1H, d, J=53.7 Hz).

MS (ESI) m/z: 424 [(M+H)⁺].

Step 2:7-[(4R)-4-fluoro-L-prolyl]-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

Trifluoroacetic acid (10 ml) was added to a dichloromethane (10 ml)solution of the compound (560 mg, 1.32 mmol) obtained in Step 1 aboveand the resulting mixture was stirred at room temperature for 1 hour.The reaction solvent was concentrated under reduced pressure and thensaturated aqueous sodium bicarbonate solution (30 ml) was added,followed by extraction with chloroform three times. The organic layerwas washed with saturated brine and dried over anhydrous sodium sulfate.The solvent was evaporated under reduced pressure to give the titlecompound (380 mg, 89%) as a pale yellow solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.98-1.11 (4H, m), 2.00-2.07 (1H,m), 2.10-2.16 (1H, m), 2.68-2.77 (1H, m), 3.04-3.14 (1H, m), 3.51 (2H,s), 3.63-3.78 (4H, m), 4.05-4.13 (1H, m), 5.23 (1H, d, J=53.5 Hz).

MS (ESI) m/z: 324 [(M+H)⁺].

Reference Example 4

Step 1: methylN-benzyl-N-[(1-{[(benzyloxy)carbonyl]amino}cyclopropyl)carbonyl]-L-alaninate

N,N′-dicyclohexylcarbodiimide (6.0 g, 29.0 mmol) was added to adichloromethane (150 ml) solution of methyl N-benzyl-L-alaninate (5.20g, 26.9 mmol) and 1-{[(benzyloxy)carbonyl]amino}cyclopropanecarboxylicacid (6.30 g, 26.7 mmol) under ice cooling and the resulting mixture wasstirred overnight at room temperature. The reaction mixture wasconcentrated under reduced pressure and then diluted with ethyl acetate,insoluble matter was removed by filtration and then the filtrate waswashed with 1 N aqueous hydrochloric acid solution and saturated brineand dried over anhydrous sodium sulfate. The solvent was evaporatedunder reduced pressure and the obtained residue was purified by silicagel column chromatography [n-hexane:ethyl acetate=1:3 (v/v)] to give thetitle compound (7.80 g, 71%) as a colorless oil.

¹H-NMR (400 MHz, CDCl₃, 70° C.) δ: 1.03-1.11 (2H, m), 1.37 (3H, d, J=7.1Hz), 1.43-1.47 (2H, m), 3.72 (3H, s), 4.48 (1H, brs), 4.61 (1H, d,J=16.6 Hz), 4.91 (1H, d, J=16.6 Hz), 4.99 (2H, s), 5.20 (1H, brs),7.20-7.34 (10H, m).

Step 2: (6S)-7-benzyl-6-methyl-4,7-diazaspiro[2.5]octane-5,8-dione

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 2 to give the title compound as a colorlessoil.

¹H-NMR (400 MHz, CDCl₃) δ: 0.98-1.07 (2H, m), 1.36 (1H, m), 1.47 (3H, d,J=7.1 Hz), 1.85 (1H, m), 3.91 (1H, q, J=7.1 Hz), 4.06 (1H, d, J=14.9Hz), 5.21 (1H, d, J=14.9 Hz), 7.26-7.37 (5H, m).

Step 3: (6S)-7-benzyl-6-methyl-4,7-diazaspiro[2.5]octane

The compound obtained in Step 2 above was reacted in the same way as inStep 3 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.30-0.34 (1H, m), 0.36-0.40 (1H, m),0.48-0.53 (1H, m), 0.55-0.61 (1H, m), 1.16 (3H, d, J=6.3 Hz), 2.12 (1H,d, J=11.7 Hz), 2.26-2.33 (2H, m), 2.74 (1H, dd, J=13.2, 9.3 Hz), 2.90(1H, dd, J=13.2, 3.4 Hz), 3.15 (1H, d, J=13.4 Hz), 4.07 (1H, d, J=13.4Hz), 7.20-7.34 (6H, m).

Step 4:(6S)-7-benzyl-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 3 above was reacted in the same way as inStep 4 of Reference Example 2 to give the title compound as a colorlessoil.

¹H-NMR (400 MHz, CDCl₃) δ: 0.68 (1H, m), 0.79 (1H, m), 0.93 (1H, m),1.21 (3H, d, J=6.1 Hz), 1.33 (1H, m), 2.17 (1H, d, J=12.0 Hz), 2.49 (1H,d, J=12.0 Hz), 2.59 (1H, m), 3.16 (1H, d, J=13.4 Hz), 3.33 (1H, dd,J=13.4, 9.3 Hz), 3.77 (1H, d, J=13.4 Hz), 4.04 (1H, d, J=13.4 Hz),7.23-7.34 (5H, m).

Step 5: (6S)-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octanehydrochloride

The compound obtained in Step 4 above was reacted in the same way as inStep 5 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.95 (1H, brs), 1.12 (1H, brs), 1.27 (1H,m), 1.33 (3H, d, J=6.3 Hz), 1.41 (1H, brs), 2.90 (1H, m), 3.38-3.59 (3H,m), 4.03 (1H, brs).

MS (ESI) m/z: 223 [(M+H)⁺].

Reference Example 5

Step 1: tert-butyl(2S,4R)-4-fluoro-2-{[(6S)-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]oct-7-yl]carbonyl}pyrrolidine-1-carboxylate

The compound obtained in Step 5 of Reference Example 4 instead of thecompound obtained in Step 5 of Reference Example 2 was reacted in thesame way as in Step 1 of Reference Example 3 to give the title compoundas a pale yellow oil.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.69-0.71 (2H, m), 1.11 (3H, d,J=6.6 Hz), 1.28-1.32 (2H, m), 1.37 (9H, s), 1.49 (1H, m), 2.05 (1H, m),3.49-3.68 (5H, m), 3.88 (1H, brs), 4.67-4.75 (2H, m), 5.27 (1H, d,J=54.0 Hz).

MS (ESI) m/z: 460 [(M+Na)⁺].

Step 2:(6S)-7-[(4R)-4-fluoro-L-prolyl]-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 3 to give the title compound as a paleyellow oil.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.68-0.77 (2H, m), 1.12 (3H, d,J=6.6 Hz), 1.28 (1H, dd, J=17.0, 7.1 Hz), 1.49 (1H, dd, J=17.0, 7.1 Hz),2.01-2.24 (2H, m), 2.65-2.68 (1H, m), 3.00-3.14 (1H, m), 3.42-3.61 (3H,m), 3.90 (1H, brs), 4.08 (1H, t, J=7.6 Hz), 4.66-4.69 (1H, m), 5.25 (1H,dt, J=55.6, 4.5 Hz).

Reference Example 6

Step 1: methylN-benzyl-N-[(1-{[(benzyloxy)carbonyl]amino}cyclopropyl)carbonyl]-D-alaninate

Methyl N-benzyl-D-alaninate instead of methyl N-benzyl-L-alaninate wasreacted in the same way as in Step 1 of Reference Example 4 to give thetitle compound as a colorless oil.

¹H-NMR (400 MHz, CDCl₃) δ: 1.07 (2H, brs), 1.36 (3H, d, J=7.1 Hz), 1.43(2H, brs), 3.61 (3H, s), 4.48-4.52 (1H, m), 4.61 (1H, d, J=17.4 Hz),4.87 (1H, d, J=17.4 Hz), 4.98 (2H, s), 5.12 (1H, brs), 7.20-7.32 (10H,m).

MS (ESI) m/z: 411 [(M+H)⁺].

Step 2: (6R)-7-benzyl-6-methyl-4,7-diazaspiro[2.5]octane-5,8-dione

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 2 to give the title compound as a colorlessoil.

¹H-NMR (400 MHz, CDCl₃) δ: 0.97-1.03 (2H, m), 1.34-1.41 (2H, m), 1.47(3H, d, J=7.1 Hz), 1.81-1.88 (1H, m), 4.06 (1H, d, J=14.9 Hz), 5.19 (1H,d, J=14.9 Hz), 6.74 (1H, brs), 7.25-7.37 (5H, m).

Step 3: (6R)-7-benzyl-6-methyl-4,7-diazaspiro[2.5]octane

The compound obtained in Step 2 above was reacted in the same way as inStep 3 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.30-0.40 (2H, m), 0.48-0.61 (2H, m), 1.16(3H, d, J=6.3 Hz), 1.58 (1H, brs), 2.12 (1H, d, J=11.5 Hz), 2.25-2.33(2H, m), 2.73 (1H, dd, J=13.1, 9.3 Hz), 2.90 (1H, dd, J=13.1, 3.0 Hz),3.15 (1H, d, J=13.4 Hz), 4.08 (1H, d, J=13.4 Hz), 7.20-7.33 (5H, m).

Step 4:(6R)-7-benzyl-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 3 above was reacted in the same way as inStep 4 of Reference Example 2 to give the title compound as a colorlessoil.

¹H-NMR (400 MHz, CDCl₃) δ: 0.64-0.92 (3H, m), 1.20 (3H, d, J=6.1 Hz),1.30-1.36 (1H, m), 2.16 (1H, d, J=12.0 Hz), 2.48 (1H, d, J=12.0 Hz),2.58 (1H, brs), 3.15 (1H, d, J=13.4 Hz), 3.31 (1H, dd, J=13.4, 9.5 Hz),3.76 (1H, d, J=13.4 Hz), 4.02 (1H, d, J=13.4 Hz), 7.23-7.33 (5H, m).

Step 5: (6R)-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octanehydrochloride

The compound obtained in Step 4 above was reacted in the same way as inStep 5 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, DMSO-d₆, 70° C.) δ: 0.93 (1H, brs), 1.17 (1H, brs),1.24-1.30 (1H, m), 1.34 (3H, d, J=6.3 Hz), 1.40 (1H, brs), 2.89 (1H, d,J=12.0 Hz), 3.14 (1H, brs), 3.40-3.46 (2H, m), 4.04 (1H, brs), 9.84 (2H,brs).

MS (ESI) m/z: 223 [(M+H)⁺].

Reference Example 7

Step 1: tert-butyl(2S,4R)-4-fluoro-2-{[(6R)-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]oct-7-yl]carbonyl}pyrrolidine-1-carboxylate

The compound obtained in Step 5 of Reference Example 6 instead of thecompound obtained in Step 5 of Reference Example 2 was reacted in thesame way as in Step 1 of Reference Example 3 to give the title compoundas a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.68-0.71 (2H, m), 1.09 (3H, brs),1.28-1.31 (2H, m), 1.36 (9H, s), 1.47 (1H, m), 1.95 (1H, brs), 3.46-3.68(5H, m), 3.91 (1H, brs), 4.69-4.71 (2H, m), 5.23 (1H, d, J=54.6 Hz).

Step 2:(6R)-7-[(4R)-4-fluoro-L-prolyl]-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 3 to give the title compound as a paleyellow oil.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.67-0.73 (1H, m), 0.86 (1H, brs),1.12 (3H, d, J=5.9 Hz), 1.28 (1H, dt, J=12.4, 5.0 Hz), 1.49 (1H, dt,J=12.4, 5.2 Hz), 1.94-2.22 (2H, m), 2.65-2.68 (1H, m), 3.13 (1H, ddd,J=32.5, 13.0, 4.2 Hz), 3.43-3.61 (3H, m), 3.92 (1H, brs), 4.04-4.06 (1H,m), 4.65 (1H, brs), 5.24 (1H, dt, J=55.6, 4.6 Hz).

Reference Example 8

Step 1: methyl(2S)-2-{benzyl[(1-{[(benzyloxy)carbonyl]amino}cyclopropyl)carbonyl]amino}butanoate

Methyl (2S)-2-(benzylamino)butanoate instead of methylN-benzyl-L-alaninate was reacted in the same way as in Step 1 ofReference Example 4 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.70 (3H, t, J=7.4 Hz), 0.83-0.89(1H, m), 1.00-1.06 (1H, m), 1.09-1.16 (1H, m), 1.30-1.37 (1H, m),1.58-1.68 (1H, m), 1.83-1.92 (1H, m), 2.87-2.92 (1H, m), 3.48 (3H, s),4.50-4.56 (1H, m), 4.64 (1H, d, J=15.9 Hz), 5.00 (2H, q, J=12.1 Hz),7.17-7.33 (10H, m), 7.76 (1H, brs).

MS (ESI) m/z: 425 [(M+H)⁺].

Step 2: (6S)-7-benzyl-6-ethyl-4,7-diazaspiro[2.5]octane-5,8-dione

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.93-0.98 (2H, m), 0.99 (3H, t, J=7.4 Hz),1.35-1.40 (1H, m), 1.79-1.86 (1H, m), 1.91-1.97 (2H, m), 3.90 (1H, t,J=5.4 Hz), 3.94 (1H, d, J=14.9 Hz), 5.35 (1H, d, J=14.9 Hz), 7.25-7.36(5H, m), 7.39 (1H, brs).

MS (ESI) m/z: 259 [(M+H)⁺].

Step 3:(6S)-7-benzyl-6-ethyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 2 above was reacted in the same way as inStep 3 of Reference Example 2 and then the compound obtained was reactedin the same way as in Step 4 of Reference Example 2 to give the titlecompound as a colorless oil.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.65-0.70 (1H, m), 0.85-0.90 (2H,m), 0.91 (3H, t, J=7.4 Hz), 1.18-1.23 (1H, m), 1.46-1.53 (1H, m),1.66-1.75 (1H, m), 2.31-2.36 (1H, m), 2.38-2.45 (2H, m), 3.32 (1H, d,J=13.9 Hz), 3.40-3.47 (1H, m), 3.84 (1H, d, J=11.7 Hz), 3.97 (1H, d,J=13.9 Hz), 7.18-7.23 (1H, m), 7.27-7.31 (4H, m).

MS (ESI) m/z: 327 [(M+H)⁺].

Step 4: (6S)-6-ethyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octanehydrochloride

The compound obtained in Step 3 above was reacted in the same way as inStep 5 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.89-0.94 (1H, m), 0.99 (3H, t,J=7.6 Hz), 1.16-1.21 (1H, m), 1.25-1.31 (1H, m), 1.41-1.48 (1H, m),1.66-1.74 (1H, m), 1.77-1.85 (1H, m), 2.86 (1H, d, J=12.9 Hz), 3.24-3.32(1H, m), 3.37-3.44 (1H, m), 3.45 (1H, d, J=12.9 Hz), 4.06-4.14 (1H, m).

MS (ESI) m/z: 237 [(M+H)⁺].

Reference Example 9

Step 1: tert-butyl(2S,4R)-2-{[(6S)-6-ethyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]oct-7-yl]carbonyl}-4-fluoropyrrolidine-1-carboxylate

1-chloro-N,N,2-trimethyl-1-propenylamine (0.35 ml, 2.68 mmol) was addedto a dichloromethane (8 ml) solution of(4R)-1-(tert-butoxycarbonyl)-4-fluoro-L-proline (520 mg, 2.23 mmol)under ice cooling. The resulting mixture was stirred at the sametemperature for 1 hour, then the compound (730 mg, 2.68 mmol) obtainedin Step 4 of Reference Example 8 and triethylamine (0.78 ml, 5.58 mmol)were added and the resulting mixture was further stirred at roomtemperature for 3 hours. The reaction mixture was diluted withchloroform and the organic layer was washed with saturated aqueoussodium bicarbonate solution and saturated brine and dried over anhydroussodium sulfate. The solvent was evaporated under reduced pressure andthe residue obtained was purified by silica gel column chromatography[n-hexane:ethyl acetate=2:1 (v/v)] to give the title compound (780 mg,77%) as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.67-0.73 (2H, m), 0.83-0.88 (3H,m), 1.29-1.33 (1H, m), 1.39 (9H, s), 1.45-1.52 (3H, m), 2.07-2.17 (1H,m), 2.43-2.47 (1H, m), 3.36-3.77 (6H, m), 4.40-4.55 (1H, m), 4.70-4.86(1H, m), 5.30 (1H, d, J=54.9 Hz).

MS (ESI) m/z: 452 [(M+H)⁺].

Step 2:(6S)-6-ethyl-7-[(4R)-4-fluoro-L-prolyl]-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 3 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.66-0.75 (2H, m), 0.83 (3H, t,J=7.2 Hz), 1.24-1.31 (1H, m), 1.43-1.53 (3H, m), 1.96-2.08 (1H, m),2.16-2.32 (1H, m), 2.67-2.74 (1H, m), 2.91-2.99 (1H, m), 3.38-3.57 (4H,m), 4.05-4.16 (1H, m), 4.43-4.53 (1H, m), 5.24 (1H, d, J=56.1 Hz).

MS (ESI) m/z: 352 [(M+H)⁺].

Reference Example 10

Step 1: methyl(2R)-2-{benzyl[(1-{[(benzyloxy)carbonyl]amino}cyclopropyl)carbonyl]amino}butanoate

Methyl (2R)-2-(benzylamino)butanoate instead of methylN-benzyl-L-alaninate was reacted in the same way as in Step 1 ofReference Example 4 to give the title compound as a pale yellow solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.71 (3H, t, J=7.4 Hz), 0.85-0.90(1H, m), 1.02-1.08 (1H, m), 1.11-1.16 (1H, m), 1.32-1.38 (1H, m),1.59-1.68 (1H, m), 1.84-1.93 (1H, m), 3.49 (3H, s), 4.30-4.38 (1H, m),4.49-4.57 (1H, m), 4.65 (1H, d, J=16.1 Hz), 5.01 (2H, q, J=12.2 Hz),7.18-7.34 (10H, m), 7.77 (1H, brs).

MS (ESI) m/z: 425 [(M+H)⁺].

Step 2: (6R)-7-benzyl-6-ethyl-4,7-diazaspiro[2.5]octane-5,8-dione

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.93-0.98 (2H, m), 0.99 (3H, t, J=7.6 Hz),1.35-1.40 (1H, m), 1.80-1.86 (1H, m), 1.91-1.98 (2H, m), 3.89 (1H, t,J=5.2 Hz), 3.94 (1H, d, J=14.9 Hz), 5.35 (1H, d, J=14.9 Hz), 7.25-7.35(5H, m), 7.51 (1H, brs).

MS (ESI) m/z: 259 [(M+1)⁺].

Step 3:(6R)-7-benzyl-6-ethyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 2 above was reacted in the same way as inStep 3 of Reference Example 2 and then the compound obtained was reactedin the same way as in Step 4 of Reference Example 2 to give the titlecompound as a colorless oil.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.65-0.70 (1H, m), 0.85-0.90 (2H,m), 0.91 (3H, t, J=7.4 Hz), 1.18-1.23 (1H, m), 1.46-1.53 (1H, m),1.66-1.75 (1H, m), 2.31-2.36 (1H, m), 2.38-2.45 (2H, m), 3.32 (1H, d,J=13.9 Hz), 3.40-3.47 (1H, m), 3.84 (1H, d, J=11.7 Hz), 3.97 (1H, d,J=13.9 Hz), 7.18-7.23 (1H, m), 7.27-7.31 (4H, m).

MS (ESI) m/z: 327 [(M+H)⁺].

Step 4: (6R)-6-ethyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octanehydrochloride

The compound obtained in Step 3 above was reacted in the same way as inStep 5 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, DMSO-d₆, 70° C.) δ: 0.89-0.94 (1H, m), 0.99 (3H, t,J=7.6 Hz), 1.16-1.21 (1H, m), 1.25-1.31 (1H, m), 1.41-1.48 (1H, m),1.66-1.74 (1H, m), 1.77-1.85 (1H, m), 2.86 (1H, d, J=12.9 Hz), 3.24-3.32(1H, m), 3.37-3.44 (1H, m), 3.45 (1H, d, J=12.9 Hz), 4.06-4.14 (1H, m).

MS (ESI) m/z: 237 [(M+H)⁺].

Reference Example 11

Step 1: tert-butyl(2S,4R)-2-{[(6R)-6-ethyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]oct-7-yl]carbonyl}-4-fluoropyrrolidine-1-carboxylate

The compound obtained in Step 4 of Reference Example 10 instead of thecompound obtained in Step 4 of Reference Example 8 was reacted in thesame way as in Step 1 of Reference Example 9 to give the title compoundas a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.69-0.73 (1H, m), 0.76-0.84 (2H,m), 0.93-0.98 (1H, m), 1.30-1.38 (3H, m), 1.39 (9H, s), 1.44-1.51 (2H,m), 1.85-2.08 (2H, m), 3.25-3.61 (4H, m), 3.66-3.77 (1H, m), 4.00-4.07(1H, m), 4.58-4.80 (2H, m), 5.25 (1H, d, J=51.3 Hz).

MS (ESI) m/z: 474 [(M+Na)⁺].

Step 2:(6R)-6-ethyl-7-[(4R)-4-fluoro-L-prolyl]-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 3 to give the title compound as a paleyellow solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.65-0.72 (1H, m), 0.79-0.85 (3H,m), 1.24-1.32 (1H, m), 1.42-1.55 (4H, m), 1.89-2.05 (1H, m), 2.12-2.20(1H, m), 2.64-2.69 (1H, m), 2.91 (1H, t, J=12.5 Hz), 3.07-3.20 (1H, m),3.38-3.58 (3H, m), 3.97-4.09 (1H, m), 4.51-4.64 (1H, m), 5.23 (1H, d,J=55.7 Hz).

MS (ESI) m/z: 352 [(M+H)⁺].

Reference Example 12

Step 1: methylN-[(1-{[(benzyloxy)carbonyl]amino}cyclopropyl)methyl]-D-leucinate

Methyl D-leucinate hydrochloride (2.73 g, 15.1 mmol), zinc chloride (2.8g, 20.5 mmol), and sodium triacetoxyborohydride (9.16 g, 41.1 mmol) wereadded to a dichloromethane (200 ml) solution of benzyl(l-formylcyclopropyl)carbamate (3.0 g, 13.7 mmol) and the resultingmixture was stirred at room temperature for 18 hours. The reactionmixture was concentrated under reduced pressure, the residue obtainedwas diluted with ethyl acetate and washed with saturated aqueous sodiumbicarbonate solution and saturated brine and then the organic layer wasdried over anhydrous magnesium sulfate. The solvent was evaporated underreduced pressure and the residue obtained was purified by silica gelcolumn chromatography [n-hexane:ethyl acetate=2:1 (v/v)] to give thetitle compound (2.24 g, 47%) as a pale yellow oil.

¹H-NMR (400 MHz, CDCl₃) δ: 0.62-0.94 (4H, m), 0.88 (3H, d, J=6.6 Hz),0.91 (3H, d, J=6.6 Hz), 1.37-1.66 (2H, m), 1.70-1.77 (1H, m), 2.46 (1H,d, J=12.9 Hz), 2.82 (1H, d, J=12.9 Hz), 3.24 (1H, t, J=7.4 Hz), 3.68(3H, s), 5.08 (1H, brs), 5.11 (2H, s), 7.22-7.42 (5H, m).

Step 2: methylN-[(1-{[(benzyloxy)carbonyl]amino}cyclopropyl)methyl]-N-(tert-butoxycarbonyl)-D-leucinate

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 1 to give the title compound as a colorlessoil.

¹H-NMR (400 MHz, CDCl₃) δ: 0.64-0.76 (1H, m), 0.78-0.97 (2H, m), 0.87(3H, d, J=6.6 Hz), 0.90 (3H, d, J=6.6 Hz), 1.32-1.40 (1H, m), 1.41 (9H,s), 1.42-1.67 (1H, m), 1.72-1.89 (2H, m), 3.00-3.19 (1H, m), 3.39-3.72(1H, m), 3.73 (3H, s), 3.74-4.18 (1H, m), 5.03 (1H, d, J=11.7 Hz), 5.13(1H, d, J=11.7 Hz), 5.92-6.08 (1H, m), 7.25-7.41 (5H, m).

Step 3: benzyl[1-({(tert-butoxycarbonyl)[(1R)-1-(hydroxymethyl)-3-methylbutyl]amino}methyl)cyclopropyl]carbamate

A tetrahydrofuran (30 ml) solution of the compound (1.95 g, 4.35 mmol)obtained in Step 2 above was added dropwise to a tetrahydrofuran (20 ml)suspension of lithium aluminum hydride (0.50 g, 12.2 mmol) under icecooling and the resulting mixture was stirred at the same temperaturefor 1 hour. Water and 10% aqueous citric acid solution were added toterminate the reaction. Then, the reaction mixture was diluted withethyl acetate, washed with saturated brine, and then dried overanhydrous magnesium sulfate. The solvent was evaporated under reducedpressure and the residue obtained was purified by silica gel columnchromatography [n-hexane:ethyl acetate=2:3 (v/v)] to give the titlecompound (1.26 g, 64%) as a colorless oil.

¹H-NMR (400 MHz, CDCl₃) δ: 0.69-1.01 (10H, m), 1.35-1.43 (1H, m), 1.44(9H, s), 1.45-1.53 (1H, m), 1.56-1.63 (2H, m), 3.09-4.00 (5H, m),4.98-5.13 (2H, m), 5.60 (1H, brs), 7.24-7.38 (5H, m).

Step 4: 4-benzyl 7-tert-butyl(6R)-6-isobutyl-4,7-diazaspiro[2.5]octane-4,7-dicarboxylate

Triphenylphosphine (1.57 g, 5.99 mmol) and diisopropyl azodicarboxylate(0.93 ml, 4.49 mmol) were added to a toluene (100 ml) solution of thecompound (1.26 g, 3.00 mmol) obtained in Step 3 above under ice coolingand the resulting mixture was gradually returned to room temperaturewhile being stirred for 18 hours. Further triphenylphosphine (1.57 g,5.99 mmol) and diisopropyl azodicarboxylate (0.93 ml, 4.49 mmol) wereadded and the resulting mixture was heated to reflux for 4 hours. Thereaction mixture was concentrated under reduced pressure and the residueobtained was purified by silica gel column chromatography[n-hexane:ethyl acetate=3:1 (v/v)] to give the title compound (0.57 g,47%) as a colorless oil.

¹H-NMR (400 MHz, CDCl₃, 60° C.) δ: 0.45-0.54 (1H, m), 0.61-0.71 (1H, m),0.85 (3H, d, J=6.6 Hz), 0.86 (3H, d, J=6.6 Hz), 0.96-1.05 (1H, m),1.21-1.62 (13H, m), 3.11-3.26 (2H, m), 3.42 (1H, d, J=14.0 Hz), 3.99(1H, d, J=14.0 Hz), 4.19 (1H, s), 5.12 (2H, s), 7.23-7.39 (5H, m).

Step 5: benzyl (6R)-6-isobutyl-4,7-diazaspiro[2.5]octane-4-carboxylatehydrochloride

4 N hydrochloric acid/dioxane (8 ml) was added to a dioxane (10 ml)solution of the compound (0.57 g, 1.42 mmol) obtained in Step 4 aboveunder ice cooling and the resulting mixture was gradually returned toroom temperature while being stirred for 24 hours. The reaction mixturewas concentrated under reduced pressure to give the title compound as acolorless solid.

Reference Example 13

Step 1: benzyl(6R)-7-{[(2S,4R)-1-(tert-butoxycarbonyl)-4-fluoropyrrolidin-2-yl]carbonyl}-6-isobutyl-4,7-diazaspiro[2.5]octane-4-carboxylate

The compound obtained in Step 5 of Reference Example 12 instead of thecompound obtained in Step 4 of Reference Example 8 was reacted in thesame way as in Step 1 of Reference Example 9 to give the title compoundas a pale yellow oil.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.44-0.59 (1H, m), 0.76-0.96 (7H,m), 1.04-1.58 (15H, m), 1.65-2.23 (1H, m), 2.86-3.09 (2H, m), 3.17 (1H,d, J=11.2 Hz), 3.34-3.80 (3H, m), 4.01 (1H, d, J=13.2 Hz), 4.45-4.82(1H, m), 5.03-5.39 (1H, m), 5.11 (2H, s), 7.28-7.45 (5H, m).

Step 2: tert-butyl(2S,4R)-4-fluoro-2-{[(6R)-6-isobutyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]oct-7-yl]carbonyl}pyrrolidine-1-carboxylate

5% palladium carbon (0.10 g) was added to an ethanol (20 ml) solution ofthe compound (0.65 g, 1.25 mmol) obtained in Step 1 above and theresulting mixture was subjected to catalytic reduction at roomtemperature for 18 hours in a hydrogen atmosphere. The catalyst wasremoved by filtration through celite and then the filtrate wasconcentrated under reduced pressure. The residue obtained was dissolvedin dichloromethane (20 ml), diisopropylamine (1.3 ml, 7.52 mmol) wasadded and trifluoroacetic anhydride (0.53 ml, 3.76 mmol) was added underice cooling. The resulting mixture was gradually returned to roomtemperature while being stirred for 2 hours and the reaction mixture wasconcentrated under reduced pressure. The residue obtained was dilutedwith ethyl acetate and washed with saturated aqueous sodium bicarbonatesolution, 10% aqueous citric acid solution, and saturated brine and thenthe organic layer was dried over anhydrous magnesium sulfate. Thesolvent was evaporated under reduced pressure, the residue obtained wasdissolved in dichloromethane (20 ml), trifluoroacetic acid (10 ml) wasadded under ice cooling and the resulting mixture was stirred at roomtemperature for 2 hours. The reaction mixture was concentrated underreduced pressure, then the residue obtained was diluted with chloroformand washed with saturated aqueous sodium bicarbonate solution andsaturated brine and then the organic layer was dried over anhydrousmagnesium sulfate. The solvent was evaporated under reduced pressure andthe residue obtained was purified by silica gel column chromatography[ethyl acetate:ethanol=4:1 (v/v)] to give the title compound (0.28 g,59%) as a pale yellow oil.

MS (ESI) m/z: 380 [(M+H)⁺].

Reference Example 14

4,9-diazadispiro[2.2.2.2]decane dihydrochloride

A borane-tetrahydrofuran complex (1.02 M tetrahydrofuran solution) (41ml, 41.6 mmol) was added dropwise to a tetrahydrofuran (20 ml) solutionof 4,9-diazadispiro[2.2.2.2]decane-5,10-dione (1.70 g, 10.4 mmol) underice cooling and then the resulting mixture was heated to reflux for 18.5hours. Methanol (50 ml) was added to the reaction mixture under icecooling, the resulting mixture was stirred for 60 minutes and then thesolvent was concentrated under reduced pressure. Ethanol (50 ml), water(25 ml), and triethylamine (25 ml) were added to the residue obtained,the resulting mixture was heated to reflux for 4 hours and then thesolvent was concentrated under reduced pressure. The residue obtainedwas dissolved in tetrahydrofuran (20 ml), triethylamine (4.3 ml, 31.2mmol) and di-tert-butyl dicarbonate (6.80 g, 31.2 mmol) were added underice cooling and the resulting mixture was stirred at room temperaturefor 14 hours. The solvent was concentrated under reduced pressure andthe residue obtained was diluted with ethyl acetate, washed with aqueoussolution of saturated ammonium chloride and saturated brine, and thendried over anhydrous sodium sulfate. The solvent was evaporated underreduced pressure, trifluoroacetic acid (20 ml) was added to a chloroform(20 ml) solution of the residue obtained and the resulting mixture wasstirred at room temperature for 1 hour. The solvent was concentratedunder reduced pressure, 4 N hydrochloric acid/dioxane (50 ml) was addedto the residue obtained and the resulting mixture was stirred for 2hours. The solvent was evaporated under reduced pressure, diethyl etherwas added to the residue and the deposited solid was collected byfiltration to give the title compound (2.00 g, 91%) as a colorlesssolid.

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.02 (4H, t, J=6.5 Hz), 1.24 (4H, t, J=6.3Hz), 3.41 (4H, s), 10.30 (2H, s).

MS (ESI) m/z: 212 [(M+H)⁺].

Reference Example 15

Step 1: tert-butyl(2S,4R)-4-fluoro-2-{[9-(trifluoroacetyl)-4,9-diazadispiro[2.2.2.2]decan-4-yl]carbonyl}pyrrolidine-1-carboxylate

Diisopropylethylamine (1.15 ml, 6.60 mmol) and(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate(1.14 g, 2.20 mmol) were added to a dimethylformamide (10 ml) solutionof (4R)-1-(tert-butoxycarbonyl)-4-fluoro-L-proline (466 mg, 2.00 mmol)and the compound (422 mg, 2.00 mmol) obtained in Reference Example 14under ice cooling. The resulting mixture was stirred at room temperaturefor 19 hours and then the reaction mixture was diluted with ethylacetate, washed with an aqueous solution of saturated ammonium chloride,saturated aqueous sodium bicarbonate solution, and saturated brine, andthen dried over anhydrous sodium sulfate. The solvent was evaporatedunder reduced pressure, then the residue obtained was dissolved indichloromethane (10 ml) and triethylamine (0.56 ml, 4.00 mmol) wasadded. After ice cooling, trifluoroacetic anhydride (0.28 ml, 2.00 mmol)was added. The resulting mixture was stirred at room temperature for 7hours and then the reaction mixture was concentrated under reducedpressure, diluted with ethyl acetate, washed with an aqueous solution ofsaturated ammonium chloride and saturated brine, and then dried overanhydrous sodium sulfate. The solvent was evaporated under reducedpressure and the residue was purified by silica gel columnchromatography [n-hexane:ethyl acetate=1:1 (v/v)] to give the titlecompound (349 mg, 39%) as a pale yellow oil.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 1.15-1.33 (18H, m), 2.04 (1H, dt,J=35.5, 7.0 Hz), 2.53 (1H, s), 3.59-3.69 (5H, m), 4.97 (1H, s), 5.31(1H, d, J=54.2 Hz).

MS (ESI) m/z: 472 [(M+Na)⁺].

Step 2:4-[(4R)-4-fluoro-L-prolyl]-9-(trifluoroacetyl)-4,9-diazadispiro[2.2.2.2]decane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 3 to give the title compound as a paleyellow solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.08-1.23 (8H, m), 2.13 (1H, s), 2.63 (1H,s), 3.67-3.73 (5H, m), 5.16 (1H, s), 5.41 (1H, d, J=53.5 Hz), 5.91 (2H,s).

MS (ESI) m/z: 350 [(M+H)⁺].

Reference Example 16

Step 1: tert-butyl(2S)-4,4-difluoro-2-{[(6S)-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]oct-7-yl]carbonyl}pyrrolidine-1-carboxylate

1-(tert-butoxycarbonyl)-4,4-difluoro-L-proline instead of(4R)-1-(tert-butoxycarbonyl)-4-fluoro-L-proline and the compoundobtained in Step 5 of Reference Example 4 instead of the compoundobtained in Step 5 of Reference Example 2 were reacted in the same wayas in Step 1 of Reference Example 3 to give the title compound as acolorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 80° C.) δ: 0.72 (2H, brs), 1.11 (3H, brs),1.25-1.29 (2H, m), 1.38 (9H, s), 1.48 (1H, brs), 2.21-2.33 (1H, m), 2.87(1H, brs), 3.02-3.10 (2H, m), 3.54 (2H, brs), 3.65-3.75 (1H, m),3.79-3.87 (1H, m), 4.64 (1H, brs).

MS (ESI) m/z: 456 [(M+H)⁺].

Step 2:(6S)-7-(4,4-difluoro-L-prolyl)-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 3 to give the title compound as a colorlessoil.

¹H-NMR (400 MHz, DMSO-d₆, 80° C.) δ: 0.70-0.74 (2H, m), 1.11 (3H, d,J=6.6 Hz), 1.23-1.29 (1H, m), 1.45-1.51 (1H, m), 2.32-2.45 (2H, m),2.92-3.08 (2H, m), 3.16-3.26 (1H, m), 3.56 (3H, brs), 3.88 (1H, brs),4.02-4.07 (1H, m), 4.64 (1H, brs).

MS (ESI) m/z: 356 [(M+H)⁺].

Reference Example 17

Step 1: tert-butyl(2S)-4,4-difluoro-2-{[(6R)-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]-oct-7-yl]carbonyl}pyrrolidine-1-carboxylate

1-(tert-butoxycarbonyl)-4,4-difluoro-L-proline instead of(4R)-1-(tert-butoxycarbonyl)-4-fluoro-L-proline and the compoundobtained in Step 5 of Reference Example 6 instead of the compoundobtained in Step 5 of Reference Example 2 were reacted in the same wayas in Step 1 of Reference Example 3 to give the title compound as acolorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.72 (2H, brs), 1.11 (3H, brs),1.25-1.31 (2H, m), 1.38 (9H, s), 1.46-1.52 (2H, m), 2.07 (1H, brs),2.82-2.92 (1H, m), 3.53 (2H, brs), 3.64-3.86 (3H, m), 4.82 (1H, brs).

MS (ESI) m/z: 478 [(M+Na)⁺].

Step 2:(6R)-7-(4,4-difluoro-L-prolyl)-6-methyl-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 3 to give the title compound as a colorlessoil.

¹H-NMR (400 MHz, DMSO-d₆, 100° C.) δ: 0.66-0.83 (2H, m), 1.10 (3H, d,J=6.5 Hz), 1.22-1.28 (1H, m), 1.44-1.51 (1H, m), 2.23-2.46 (2H, m),2.92-3.01 (2H, m), 3.22-3.26 (1H, m), 3.43-3.60 (3H, m), 4.14 (1H, brs),4.61 (1H, brs).

MS (ESI) m/z: 356 [(M+H)⁺].

Reference Example 18

Step 1: methyl N-benzyl-O-tert-butylserinate

Triethylamine (9.5 ml, 68.0 mmol) and benzaldehyde (6.37 ml, 63.0 mmol)were added to a methanol (300 ml) solution of methylO-tert-butyl-L-serinate hydrochloride (12.0 g, 57.0 mmol) and theresulting mixture was stirred at room temperature for 6 hours. Sodiumborohydride (3.23 g, 86.0 mmol) was gradually added under ice coolingand then the resulting mixture was stirred at room temperature for 2hours. An aqueous solution of saturated ammonium chloride (100 ml) wasadded to the reaction mixture. After concentration under reducedpressure, the residue was diluted with ethyl acetate and theprecipitated insoluble matter was removed by filtration. The organiclayer was washed with saturated brine and then dried over anhydroussodium sulfate. The solvent was evaporated under reduced pressure togive the title compound (15.3 g, 100%) as a colorless oil.

¹H-NMR (400 MHz, CDCl₃) δ: 1.14 (9H, s), 2.09 (1H, brs), 3.44 (1H, t,J=5.0 Hz), 3.55-3.63 (2H, m), 3.70-3.73 (1H, m), 3.72 (3H, s), 3.91 (1H,d, J=13.2 Hz), 7.23-7.38 (5H, m).

MS (ESI) m/z: 266 [(M+H)⁺].

Step 2: methylN-benzyl-N-[(1-{[(benzyloxy)carbonyl]amino}cyclopropyl)carbonyl]-O-tert-butylserinate

The compound obtained in Step 1 above instead of methylN-benzyl-L-alaninate was reacted in the same way as in Step 1 ofReference Example 4 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.94-0.97 (2H, m), 0.98 (9H, s),1.16-1.24 (2H, m), 3.53 (3H, s), 3.63-3.72 (2H, m), 4.46-4.53 (1H, m),4.60-4.70 (1H, m), 4.83 (1H, d, J=17.3 Hz), 4.94-5.01 (2H, m), 7.16-7.35(10H, m), 7.66 (1H, brs).

MS (ESI) m/z: 483 [(M+H)⁺].

Step 3:7-benzyl-6-(tert-butoxymethyl)-4,7-diazaspiro[2.5]octane-5,8-dione

The compound obtained in Step 2 above was reacted in the same way as inStep 2 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃, 50° C.) δ: 0.88-0.97 (2H, m), 1.15 (9H, s),1.36-1.41 (1H, m), 1.77-1.83 (1H, m), 3.69 (2H, ddd, J=20.5, 9.6, 3.1Hz), 3.96 (1H, t, J=3.1 Hz), 4.07 (1H, d, J=15.1 Hz), 5.29 (1H, d,J=14.9 Hz), 6.64 (1H, brs), 7.24-7.34 (5H, m).

MS (ESI) m/z: 261 [(M-tBu)⁺].

Step 4: 7-benzyl-6-(hydroxymethyl)-4,7-diazaspiro[2.5]octane-5,8-dione

Trifluoroacetic acid (30 ml) was added to a chloroform (50 ml) solutionof the compound (10.5 g, 33.0 mmol) obtained in Step 3 above and theresulting mixture was stirred at room temperature for 1 hour and thenfurther stirred under heating at 40° C. for 6 hours. The solvent wasconcentrated under reduced pressure, then the reaction mixture wassubjected to azeotropic distillation with toluene and the residue wasdiluted with ethyl acetate, then washed with saturated aqueous sodiumbicarbonate solution and saturated brine, and dried over anhydrousmagnesium sulfate. The solvent was evaporated under reduced pressure andthe residue obtained was purified by silica gel column chromatography[chloroform:methanol=15:1 (v/v)] to give the title compound (7.15 g,83%) as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.96-1.06 (2H, m), 1.36-1.42 (1H, m),1.74-1.80 (1H, m), 2.98-3.05 (1H, m), 3.91 (1H, s), 3.91-4.00 (1H, m),4.11 (1H, d, J=15.1 Hz), 5.28 (1H, d, J=15.1 Hz), 7.25-7.42 (6H, m).

MS (ESI) m/z: 261 [(M+H)⁺].

Step 5:7-benzyl-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4,7-diazaspiro[2.5]octane-5,8-dione

4-dimethylaminopyridine (660 mg, 5.00 mmol) andtert-butyldimethylchlorosilane (4.90 g, 32.0 mmol) were added to adimethylformamide (100 ml) solution of the compound (7.15 g, 27.0 mmol)obtained in Step 4 above and triethylamine (4.5 ml, 32.0 mmol). Theresulting mixture was stirred at room temperature for 16 hours, then icewater (200 ml) was added to the reaction mixture and the precipitatedsolid was collected by filtration. The solid obtained was purified bysilica gel column chromatography [n-hexane:ethyl acetate=3:1 (v/v)] togive the title compound (8.63 g, 85%) as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.04 (3H, s), 0.05 (3H, s), 0.89 (9H, s),0.91-1.00 (2H, m), 1.40-1.46 (1H, m), 1.74-1.81 (1H, m), 3.90-3.94 (2H,m), 4.00 (1H, dd, J=11.1, 3.5 Hz), 4.08 (1H, d, J=15.1 Hz), 5.32 (1H, d,J=15.1 Hz), 7.25-7.38 (5H, m).

MS (ESI) m/z: 375 [(M+H)⁺].

Step 6:7-benzyl-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 5 above was reacted in the same way as inStep 3 of Reference Example 2 to give the title compound as a colorlessoil.

¹H-NMR (400 MHz, CDCl₃) δ: 0.05 (3H, s), 0.06 (3H, s), 0.31-0.38 (2H,m), 0.51-0.59 (2H, m), 0.89 (9H, s), 2.23 (1H, d, J=11.7 Hz), 2.30 (1H,d, J=11.7 Hz), 2.40-2.45 (1H, m), 2.81 (1H, dd, J=13.2, 8.3 Hz), 3.16(1H, dd, J=13.2, 3.4 Hz), 3.34 (1H, d, J=13.9 Hz), 3.65 (1H, dd, J=10.4,6.5 Hz), 3.96 (1H, dd, J=10.3, 4.4 Hz), 4.10 (1H, d, J=13.7 Hz),7.21-7.37 (5H, m).

MS (ESI) m/z: 347 [(M+H)⁺].

Step 7:7-benzyl-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 6 above was reacted in the same way as inStep 4 of Reference Example 2 to give the title compound as a paleyellow oil.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.00 (3H, s), 0.01 (3H, s),0.61-0.68 (1H, m), 0.84 (9H, s), 0.85-0.90 (2H, m), 1.18-1.25 (1H, m),2.28 (1H, d, J=11.7 Hz), 2.45-2.49 (1H, m), 2.54-2.58 (1H, m), 3.34-3.41(2H, m), 3.50 (1H, dd, J=10.3, 7.8 Hz), 3.91 (1H, dd, J=11.5, 5.1 Hz),3.95 (1H, d, J=14.2 Hz), 4.03 (1H, d, J=14.2 Hz), 7.16-7.20 (1H, m),7.24-7.28 (4H, m).

MS (ESI) m/z: 443 [(M+H)⁺].

Step 8:6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 7 above was reacted in the same way as inStep 5 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.08 (6H, s), 0.76-0.80 (1H, m),0.90 (9H, s), 0.91-0.94 (1H, m), 1.10-1.16 (1H, m), 1.36-1.42 (1H, m),2.54 (1H, d, J=12.9 Hz), 3.04-3.09 (1H, m), 3.22-3.29 (2H, m), 3.66 (1H,dd, J=10.4, 7.0 Hz), 3.74 (1H, dd, J=10.5, 4.9 Hz), 4.04-4.12 (1H, m).

MS (ESI) m/z: 353 [(M+H)⁺].

Step 9: tert-butyl(2S,4R)-2-{[(6R)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]oct-7-yl]carbonyl}-4-fluoropyrrolidine-1-carboxylate(isomer A) and tert-butyl(2S,4R)-2-{[(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]oct-7-yl]carbonyl}-4-fluoropyrrolidine-1-carboxylate(isomer B)

The compound obtained in Step 8 above instead of the compound obtainedin Step 4 of Reference Example 8 was reacted in the same way as in Step1 of Reference Example 9 and then the mixture of diastereomers obtainedwas resolved by silica gel column chromatography [n-hexane:ethylacetate=3:1 (v/v)] to respectively give the title compounds as acolorless solid.

Isomer A: ¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.00 (3H, s), 0.01 (3H,s), 0.68-0.76 (1H, m), 0.84 (9H, s), 0.85-0.87 (1H, m), 1.23-1.30 (1H,m), 1.35 (9H, s), 1.40-1.45 (1H, m), 1.99-2.16 (1H, m), 2.31-2.49 (1H,m), 3.33-3.77 (7H, m), 4.12-4.28 (1H, m), 4.34-4.48 (1H, m), 4.60-4.76(1H, m), 5.26 (1H, d, J=52.7 Hz).

MS (ESI) m/z: 590 [(M+Na)⁺].

Isomer B: ¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: −0.01 (3H, s), 0.01 (3H,s), 0.66-0.75 (1H, m), 0.83 (9H, s), 1.26-1.34 (3H, m), 1.35 (9H, s),1.74-1.96 (2H, m), 3.27-3.73 (7H, m), 4.12-4.28 (1H, m), 4.38-4.52 (1H,m), 4.68-4.78 (1H, m), 5.20 (1H, d, J=55.2 Hz).

MS (ESI) m/z: 590 [(M+Na)⁺].

Reference Example 19

(6R)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-7-[(4R)-4-fluoro-L-prolyl]-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound (isomer A) obtained in Step 9 of Reference Example 18 wasreacted in the same way as in Step 2 of Reference Example 3 to give thetitle compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.01 (6H, s), 0.66-0.79 (2H, m),0.83 (9H, s), 1.20-1.31 (1H, m), 1.37-1.47 (1H, m), 1.92-2.07 (1H, m),2.09-2.26 (1H, m), 2.80-2.92 (2H, m), 3.39-3.71 (6H, m), 3.98-4.18 (1H,m), 4.35-4.53 (1H, m), 5.22 (1H, d, J=55.4 Hz).

MS (ESI) m/z: 468 [(M+H)⁺].

Reference Example 20

(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-7-[(4R)-4-fluoro-L-prolyl]-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound (isomer B) obtained in Step 9 of Reference Example 18 wasreacted in the same way as in Step 2 of Reference Example 3 to give thetitle compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.01 (6H, s), 0.67-0.73 (1H, m),0.83 (9H, s), 1.20-1.28 (2H, m), 1.38-1.47 (1H, m), 1.89-2.15 (2H, m),2.68-2.80 (1H, m), 2.85-2.92 (1H, m), 3.08-3.18 (1H, m), 3.39-3.72 (4H,m), 4.02-4.19 (2H, m), 4.39-4.53 (1H, m), 5.21 (1H, d, J=55.7 Hz).

MS (ESI) m/z: 468 [(M+H)⁺].

Reference Example 21

Step 1: tert-butyl7-benzyl-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4,7-diazaspiro[2.5]octane-4-carboxylate

The compound obtained in Step 7 of Reference Example 18 was reacted inthe same way as in Step 2 of Example 1 and then reacted in the same wayas in Step 2 of Reference Example 1 to give the title compound as acolorless oil.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.00 (6H, s), 0.39-0.46 (1H, m),0.60-0.70 (2H, m), 0.84 (9H, s), 0.97-1.02 (1H, m), 1.35 (9H, s), 2.16(1H, d, J=11.7 Hz), 2.27 (1H, d, J=11.7 Hz), 3.08 (1H, dd, J=12.9, 8.3Hz), 3.32 (1H, d, J=14.2 Hz), 3.49 (1H, dd, J=10.5, 6.6 Hz), 3.79-3.88(2H, m), 3.95 (1H, d, J=14.2 Hz), 7.13-7.16 (1H, m), 7.21-7.27 (4H, m).

MS (ESI) m/z: 447 [(M+1)]⁺.

Step 2: tert-butyl6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4,7-diazaspiro[2.5]octane-4-carboxylate

The compound obtained in Step 1 above was reacted in the same way as inStep 5 of Reference Example 2 to give the title compound as a paleyellow oil.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.06 (6H, s), 0.48-0.57 (2H, m),0.78-0.83 (1H, m), 0.89 (9H, s), 1.14-1.20 (1H, m), 1.40 (9H, s), 2.16(1H, d, J=12.5 Hz), 2.62-2.68 (1H, m), 2.75 (1H, dd, J=12.5, 10.0 Hz),2.96 (1H, dd, J=12.5, 2.0 Hz), 3.47 (1H, dd, J=10.1, 6.2 Hz), 3.54 (1H,dd, J=10.0, 5.1 Hz), 3.87 (1H, dd, J=12.5, 2.9 Hz).

MS (ESI) m/z: 357 [(M+1)]⁺.

Step 3: tert-butyl(6R)-7-({(2S,4R)-1-[(benzyloxy)carbonyl]-4-fluoropyrrolidin-2-ylcarbonyl)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4,7-diazaspiro[2.5]octane-4-carboxylate(isomer A) and tert-butyl(6S)-7-({(2S,4R)-1-[(benzyloxy)carbonyl]-4-fluoropyrrolidin-2-ylcarbonyl)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-4,7-diazaspiro[2.5]octane-4-carboxylate(isomer B)

The compound obtained in Step 2 above instead of the compound obtainedin Step 4 of Reference Example 8 was reacted in the same way as in Step1 of Reference Example 9 and then the mixture of diastereomers obtainedwas resolved by silica gel column chromatography in the same way as inStep 9 of Reference Example 18 to give the title compounds respectivelyas colorless solids.

Isomer A: ¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: −0.06 (3H, brs), 0.00(3H, s), 0.46-0.52 (1H, m), 0.82 (9H, s), 0.94-1.04 (1H, m), 1.15-1.28(2H, m), 1.36 (9H, s), 1.92-2.12 (2H, m), 2.71-2.83 (1H, m), 3.21-3.29(1H, m), 3.42-3.64 (4H, m), 3.69-3.78 (1H, m), 4.10-4.24 (1H, m),4.57-5.12 (4H, m), 5.28 (1H, d, J=52.7 Hz), 7.24-7.33 (5H, m).

MS (ESI) m/z: 628 [(M+Na)]⁺.

Isomer B: ¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: −0.01 (3H, s), 0.00 (3H,s), 0.74-0.79 (1H, m), 0.79-0.85 (1H, m), 0.82 (9H, s), 0.95-1.04 (1H,m), 1.22-1.30 (1H, m), 1.37 (9H, s), 1.80-2.01 (2H, m), 3.19-3.26 (1H,m), 3.34-3.60 (4H, m), 3.67-3.83 (2H, m), 3.98-4.13 (1H, m), 4.31-4.45(1H, m), 4.78-4.84 (1H, m), 4.87-5.10 (2H, m), 5.21, 5.28 (1H, each d,J=55.2, 54.2 Hz), 7.23-7.34 (5H, m).

MS (ESI) m/z: 628 [(M+Na)]⁺.

Reference Example 22

tert-butyl(6R)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-7-[(4R)-4-fluoro-L-prolyl]-4,7-diazaspiro[2.5]octane-4-carboxylate

The compound (isomer A) obtained in Step 3 of Reference Example 21 wasreacted in the same way as in Step 5 of Reference Example 2 to give thetitle compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.06 (6H, s), 0.49-0.59 (2H, m),0.87 (9H, s), 1.07-1.14 (1H, m), 1.31-1.39 (1H, m), 1.42 (9H, s),2.16-2.34 (2H, m), 3.00-3.43 (6H, m), 3.55-3.77 (2H, m), 3.95-4.17 (1H,m), 4.29-4.38 (1H, m), 5.37 (1H, d, J=54.4 Hz).

MS (ESI) m/z: 472 [(M+1)]⁺.

Reference Example 23

tert-butyl(6S)-6-({[tert-butyl(dimethyl)silyl]oxy}methyl)-7-[(4R)-4-fluoro-L-prolyl]-4,7-diazaspiro[2.5]octane-4-carboxylate

The compound (isomer B) obtained in Step 3 of Reference Example 21 wasreacted in the same way as in Step 5 of Reference Example 2 to give thetitle compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆, 90° C.) δ: 0.05 (6H, s), 0.51-0.58 (1H, m),0.87 (9H, s), 1.07-1.16 (1H, m), 1.30-1.40 (2H, m), 1.43 (9H, s),1.94-2.13 (2H, m), 3.03-3.20 (3H, m), 3.27-3.41 (1H, m), 3.45-3.56 (1H,m), 3.59-3.74 (1H, m), 3.90-4.11 (2H, m), 4.43-4.63 (2H, m), 5.39 (1H,d, J=53.5 Hz).

MS (ESI) m/z: 472 [(M+1)]⁺.

Reference Example 24

Step 1: methyl N-benzyl-O-tert-butyl-L-serinate

Methyl O-tert-butyl-L-serinate hydrochloride (12 g, 56.7 mmol) wasdissolved in methanol (120 ml), acetic acid (6.50 ml, 114 mmol),benzaldehyde (6.25 ml, 61.8 mmol), and sodium cyanoborohydride (1 Mtetrahydrofuran solution, 75 ml) were added and the resulting mixturewas stirred at room temperature for 48 hours. The reaction mixture wasconcentrated, diluted with water, and neutralized with sodiumbicarbonate under ice cooling, followed by extraction with chloroform.The organic layer was dried over anhydrous magnesium sulfate, then thedrying agent was removed by filtration and the solvent was concentratedunder reduced pressure. The residue obtained was purified by silica gelchromatography [ethyl acetate:n-hexane=1:4 (v/v)] to give the titlecompound (7.75 g, 52%) as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.14 (9H, s), 2.09 (1H, brs), 3.44 (1H, t,J=5.0 Hz), 3.55-3.63 (2H, m), 3.70-3.73 (1H, m), 3.72 (3H, s), 3.91 (1H,d, J=13.2 Hz), 7.23-7.38 (5H, m).

MS (ESI) m/z: 266 [(M+1)]⁺.

Step 2: methylN-benzyl-N-[(1-{[(benzyloxy)carbonyl]amino}cyclopropyl)carbonyl]-O-tert-butyl-L-serinate

The compound obtained in Step 1 above instead of methylN-benzyl-L-alaninate was reacted in the same way as in Step 1 ofReference Example 4 to give the title compound as a colorless oil.

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.94-0.98 (2H, m), 0.98 (9H, s), 1.16-1.24(2H, m), 3.53 (3H, s), 3.63-3.72 (2H, m), 4.46-4.53 (1H, m), 4.60-4.70(1H, m), 4.83 (1H, d, J=17.3 Hz), 4.94-5.01 (2H, m), 7.16-7.35 (10H, m),7.66 (1H, brs).

MS (ESI) m/z: 483 [(M+1)]⁺.

Step 3:(6S)-7-benzyl-6-(tert-butoxymethyl)-4,7-diazaspiro[2.5]octane-5,8-dione

The compound obtained in Step 2 above was reacted in the same way as inStep 2 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.88-0.97 (2H, m), 1.15 (9H, s), 1.36-1.41(1H, m), 1.77-1.83 (1H, m), 3.69 (2H, ddd, J=20.5, 9.6, 3.1 Hz), 3.96(1H, t, J=3.1 Hz), 4.07 (1H, d, J=15.1 Hz), 5.29 (1H, d, J=14.9 Hz),6.64 (1H, brs), 7.24-7.34 (5H, m).

MS (ESI) m/z: 317 [(M+1)]⁺.

Step 4: (6R)-7-benzyl-6-(tert-butoxymethyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 3 above was reacted in the same way as inStep 3 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.30-0.38 (2H, m), 0.48-0.59 (2H, m), 1.19(9H, s), 1.62 (1H, br), 2.27 (2H, AB type d, J=11.9 Hz), 2.40-2.48 (1H,m), 2.80 (1H, dd, J=13.3, 8.2 Hz), 3.14 (1H, dd, J=7.3, 3.2 Hz),3.34-3.41 (2H, m), 3.69 (1H, dd, J=9.3, 4.6 Hz), 4.12 (1H, d, J=13.7Hz), 7.21-7.35 (5H, m).

MS (ESI) m/z: 289 [(M+1)]⁺.

Step 5: [(6R)-7-benzyl-4,7-diazaspiro[2.5]oct-6-yl]methanol

The compound obtained in Step 4 above was reacted in the same way as inStep 4 of Reference Example 18 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.33-0.43 (2H, m), 0.52-0.61 (2H, m), 1.71(1H, br), 2.36-2.45 (3H, m), 3.06 (2H, d, J=6.0 Hz), 3.36 (1H, d, J=13.7Hz), 3.60 (1H, dd, J=11.0, 3.5 Hz), 3.95 (1H, dd, J=11.0, 4.5 Hz), 4.15(1H, d, J=13.7 Hz), 7.24-7.32 (5H, m).

MS (ESI) m/z: 233 [(M+1)]⁺.

Step 6: tert-butyl(6R)-7-benzyl-6-(hydroxymethyl)-4,7-diazaspiro[2.5]octane-4-carboxylate

The compound obtained in Step 5 above was reacted in the same way as inStep 2 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.44-0.49 (1H, m), 0.62-0.72 (2H,m), 1.00-1.05 (1H, m), 1.39 (9H, s), 2.18 (1H, d, J=11.7 Hz), 2.28 (1H,dd, J=11.7, 1.2 Hz), 2.41-2.46 (1H, m), 3.13 (1H, dd, J=13.2, 8.5 Hz),3.34 (1H, d, J=14.0 Hz), 3.40-3.46 (1H, m), 3.66-3.72 (1H, m), 3.82 (1H,dd, J=13.2, 3.5 Hz), 4.02 (1H, d, J=14.0 Hz), 4.14 (1H, t, J=5.4 Hz),7.16-7.30 (5H, m).

MS (ESI) m/z: 333 [(M+1)]⁺.

Step 7: tert-butyl(6R)-7-benzyl-6-(fluoromethyl)-4,7-diazaspiro[2.5]octane-4-carboxylate

A dichloromethane (8 ml) solution of the compound (360 mg, 1.08 mmol)obtained in Step 6 above was cooled to −78° C. Subsequently,bis(2-methoxyethyl)aminosulfur trifluoride (0.26 ml, 1.41 mmol) wasadded dropwise and the resulting mixture was gradually heated andstirred for 20 hours. The reaction mixture was neutralized withsaturated aqueous sodium bicarbonate solution, followed by extractionwith chloroform. The organic layer was dried over anhydrous sodiumsulfate, then the drying agent was removed by filtration and the solventwas concentrated under reduced pressure. The residue obtained waspurified by silica gel chromatography [ethyl acetate:n-hexane=1:10(v/v)] to give the title compound (325 mg, 90%) as a colorless oil.

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.60-0.67 (2H, m), 0.79-0.90 (2H,m), 1.40 (9H, s), 2.45 (1H, d, J=12.7 Hz), 2.64 (1H, d, J=12.7 Hz),2.83-2.93 (2H, m), 3.35-3.45 (1H, m), 3.71 (2H, d, J=3.2 Hz), 3.72-3.79(1H, m), 4.66-4.81 (1H, m), 7.19-7.31 (5H, m).

MS (ESI) m/z: 335 [(M+1)]⁺.

Step 8:(6R)-7-benzyl-6-(fluoromethyl)-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octane

The compound obtained in Step 7 above was reacted in the same way as inStep 5 of Reference Example 12 and then reacted in the same way as inStep 4 of Reference Example 2 to give the title compound as a colorlessoil.

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 0.84 (2H, brs), 0.92 (2H, br),2.55, 2.76 (1H, each br), 2.94 (2H, s), 2.95-2.98 (1H, m), 3.72 (2H, s),3.73-3.94 (2H, m), 4.74, 4.86 (1H, each br), 7.20-7.31 (5H, m).

Step 9:(6R)-6-(fluoromethyl)-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octanehydrochloride

The compound obtained in Step 8 above was reacted in the same way as inStep 5 of Reference Example 2 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 1.06-1.11 (4H, m), 2.65-2.90 (3H,m), 3.37-3.65 (2H, m), 3.92 (11-1, br), 5.21-5.34 (1H, m), 9.55 (2H,br).

MS (ESI) m/z: 241 [(M+1)]⁺.

Reference Example 25

Step 1: tert-butyl(2S,4R)-4-fluoro-2-{[(6R)-6-(fluoromethyl)-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]oct-7-yl]carbonyl}-4-fluoropyrrolidine-1-carboxylate

The compound obtained in Step 9 of Reference Example 24 instead of thecompound obtained in Step 4 of Reference Example 8 was reacted in thesame way as in Step 1 of Reference Example 9 to give the title compoundas a colorless solid.

MS (ESI) m/z: 456 [(M+1)]⁺.

Step 2:(6R)-6-(fluoromethyl)-7-[(4R)-4-fluoro-L-prolyl]-4-(trifluoroacetyl)-4,7-diazaspiro[2.5]octanehydrochloride

The compound obtained in Step 1 above was reacted in the same way as inStep 5 of Reference Example 12 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, d₆-DMSO, 100° C.) δ: 1.07-1.11 (4H, m), 2.04-2.31 (1H,m), 2.65-2.73 (1H, m), 3.13-3.23 (2H, m), 3.43-3.58 (2H, m), 3.62-3.75(2H, m), 4.20-4.37 (2H, m), 4.57-4.78 (1H, m), 4.92-4.98 (1H, m), 5.46(1H, d, J=53.2 Hz), 9.77 (2H, br).

MS (ESI) m/z: 356 [(M+1)]⁺.

Reference Example 26

Step 1: 3,4-bis(4-chlorophenyl)-1,2,5-thiadiazole 1,1-dioxide

1,2-bis(4-chlorophenyl)ethane-1,2-dione was used as a starting materialand reacted in the same way as in Step 7 of Reference Example 1 to givethe title compound as a pale yellow solid.

¹H-NMR (400 MHz, CDCl₃) δ: 7.47 (4H, d, J=8.8 Hz), 7.53 (4H, d, J=8.8Hz).

Step 2: 3,4-bis(4-chlorophenyl)-3-methyl-2,3-dihydro-1,2,5-thiadiazole1,1-dioxide

Methyl magnesium bromide (0.89 M tetrahydrofuran solution, 43.1 ml) wasadded dropwise to a toluene (200 ml) suspension of the compound (10.0 g,29.5 mmol) obtained in Step 1 above at 0° C. The resulting mixture wasstirred at room temperature for 1 hour and then 1 N aqueous hydrochloricacid solution was added, followed by extraction with ethyl acetate.Then, the organic layer was washed with saturated aqueous sodiumbicarbonate solution and saturated brine and dried over anhydrous sodiumsulfate and the solvent was evaporated under reduced pressure to givethe title compound (11.1 g, quantitative) as a colorless oil.

¹H-NMR (400 MHz, CDCl₃) δ: 2.06 (3H, s), 4.70 (1H, s), 7.30-7.48 (6H,m), 7.63 (2H, d, J=9.0 Hz).

Step 3: (3S*,4R*)-3,4-bis(4-chlorophenyl)-3-methyl-1,2,5-thiadiazolidine1,1-dioxide

The compound obtained in Step 2 above was reacted in the same way as inStep 8 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.85 (3H, s), 4.63 (1H, d, J=6.8 Hz), 4.72(1H, s), 4.93 (1H, d, J=6.8 Hz), 6.77 (2H, d, J=8.5 Hz), 7.01 (2H, d,J=8.5 Hz), 7.17 (2H, d, J=8.5 Hz), 7.18 (2H, d, J=8.5 Hz).

Step 4: (1R*,2S*)-1,2-bis(4-chlorophenyl)propane-1,2-diamine

The compound obtained in Step 3 above was reacted in the same way as inStep 9 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.49 (3H, s), 4.08 (1H, s), 6.98 (2H, d,J=8.5 Hz), 7.17 (2H, d, J=8.3 Hz), 7.25-7.28 (4H, m).

Step 5: (1R,2S)-1,2-bis(4-chlorophenyl)propane-1,2-diamine

The compound obtained in Step 4 above was optically resolved in the sameway as in Step 10 of Reference Example 1 to give the title compound as acolorless solid.

[α]D=±69.2° (c.1.05, methanol, 23° C.)

Step 6: (4S,5R)-4,5-bis(4-chlorophenyl)-4-methylimidazolidine-2-thione

The compound obtained in Step 5 above was reacted in the same way as inStep 11 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, DMSO-d₆) δ: 1.71 (3H, s), 4.94 (1H, s), 6.89 (2H, dt,J=8.9, 2.1 Hz), 6.97 (2H, dt, J=8.9, 2.1 Hz), 7.17-7.12 (4H, m), 8.74(1H, s), 8.92 (1H, s).

Step 7: ethyl(5R,6S)-5,6-bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole-2-carboxylate

The compound obtained in Step 6 above was reacted in the same way as inStep 12 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.03 (3H, d, J=7.0 Hz), 1.03 (3H, d, J=7.0Hz), 1.37 (3H, t, J=7.1 Hz), 2.10 (3H, s), 3.28-3.47 (1H, m), 4.33 (2H,q, J=7.1 Hz), 5.57 (1H, s), 6.45-7.18 (8H, m).

Step 8:(5R,6S)-5,6-bis(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole-2-carboxylicacid

The compound obtained in Step 7 above was reacted in the same way as inStep 13 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.83 (3H, d, J=7.1 Hz), 0.93 (3H, d, J=7.1Hz), 1.78 (3H, s), 2.99-3.67 (1H, m), 5.79 (1H, s), 6.44-7.43 (8H, m).

MS (ESI) m/z: 447 [(M+1)]⁺.

Reference Example 27

Step 1: ethyl 2-amino-2-(4-chlorophenyl)propionate

4′-chloroacetophenone instead of 1-(6-chloropyridin-3-yl)ethanone wasreacted in the same way as in Step 1 of Reference Example 1 to give thetitle compound as a yellow oil.

MS (ESI) m/z: 228 [(M+1)]⁺.

Step 2: ethyl2-[(tert-butoxycarbonyl)amino]-2-(4-chlorophenyl)propionate

The compound obtained in Step 1 above was reacted in the same way as inStep 2 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.17 (3H, t, J=7.3 Hz), 1.37 (9H, brs), 1.97(3H, s), 4.08-4.21 (2H, m), 5.92 (1H, br), 7.29-7.33 (2H, m), 7.36-7.41(2H, m).

MS (ESI) m/z: 350 [(M+Na)]⁺.

Step 3: tert-butyl [1-(4-chlorophenyl)-1-methyl-2-oxoethyl]carbamate

The compound obtained in Step 2 above was reacted in the same way as inStep 3 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.42 (9H, brs), 1.81 (3H, s), 5.71 (1H, br),7.31-7.39 (4H, m), 9.27 (1H, s).

Step 4: tert-butyl[2-(4-chloro-3-fluorophenyl)-1-(4-chlorophenyl)-2-hydroxy-1-methylethyl]carbamate

The compound obtained in Step 3 above was reacted in the same way as inStep 4 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.43 (9H, brs), 1.55 (3H, brs), 5.08 (2H,br), 5.40 (1H, br), 6.80 (1H, br), 7.02-7.07 (1H, m), 7.24-7.35 (5H, m).

MS (ESI) m/z: 436 [(M+Na)]⁺.

Step 5: tert-butyl[2-(4-chloro-3-fluorophenyl)-1-(4-chlorophenyl)-1-methyl-2-oxoethyl]carbamate

The compound obtained in Step 4 above was reacted in the same way as inStep 5 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.29 (9H, brs), 1.96 (3H, s), 6.19 (1H, br),7.28-7.42 (7H, m).

MS (ESI) m/z: 434 [(M+Na)]⁺.

Step 6:2-amino-1-(4-chloro-3-fluorophenyl)-2-(4-chlorophenyl)propan-1-one

The compound obtained in Step 5 above was reacted in the same way as inStep 6 of Reference Example 1 to give the title compound as a colorlesssolid.

MS (ESI) m/z: 312 [(M+1)]+.

Step 7:4-(4-chloro-3-fluorophenyl)-3-(4-chlorophenyl)-3-methyl-2,3-dihydro-1,2,5-thiadiazole1,1-dioxide

The compound obtained in Step 6 above was reacted in the same way as inStep 7 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 2.06 (3H, s), 4.74 (1H, br), 7.34-7.54 (7H,m).

Step 8:(3S*,4R*)-4-(4-chloro-3-fluorophenyl)-3-(4-chlorophenyl)-3-methyl-1,2,5-thiadiazole1,1-dioxide

The compound obtained in Step 7 above was reacted in the same way as inStep 8 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.78 (3H, s), 4.93 (1H, d, J=2.3 Hz), 6.95(1H, d, J=8.5 Hz), 7.07-7.14 (3H, m), 7.21 (2H, d, J=8.8 Hz), 7.35 (1H,t, J=8.1 Hz), 7.94-7.97 (2H, m).

MS (ESI) m/z: 375 [(M+1)]⁺.

Step 9:(1R*,2S*)-1-(4-chloro-3-fluorophenyl)-2-(4-chlorophenyl)propane-1,2-diamine

The compound obtained in Step 8 above was reacted in the same way as inStep 9 of Reference Example 1 to give the title compound as a colorlesssolid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.49 (3H, s), 1.50 (4H, rbs), 4.08 (1H, s),6.75 (1H, d, J=8.2 Hz), 6.94 (1H, dd, J=10.5, 1.3 Hz), 7.19 (1H, t,J=7.8 Hz), 7.26 (4H, s).

MS (ESI) m/z: 313 [(M+1)]⁺.

Step 10:(1R,2S)-1-(4-chloro-3-fluorophenyl)-2-(4-chlorophenyl)propane-1,2-diamine

The compound obtained in Step 9 above was optically resolved in the sameway as in Step 10 of Reference Example 1 to give the title compound as apale yellow oil.

MS (ESI) m/z: 313 [(M+1)]⁺.

[α]_(D)=+ 67.4° (c=1.0, chloroform, 25° C.)

Step 11:(4S,5R)-5-(4-chloro-3-fluorophenyl)-4-(4-chlorophenyl)-4-methylimidazolidine-2-thione

The compound obtained in Step 10 above was reacted in the same way as inStep 11 of Reference Example 1 to give the title compound as a paleyellow oil. This compound was used in next reaction without beingpurified.

Step 12: ethyl(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole-2-carboxylate

The compound obtained in Step 11 above was reacted in the same way as inStep 12 of Reference Example 1 to give the title compound as a paleyellow solid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.90 (3H, d, J=7.3 Hz), 1.02 (3H, d, J=7.1Hz), 1.34 (3H, t, J=7.2 Hz), 1.80 (3H, s), 3.35-3.37 (1H, m), 4.25 (2H,q, J=7.1 Hz), 5.03 (1H, s), 6.54 (2H, brs), 7.07-7.12 (5H, m).

Step 13:(5R,6S)-5-(4-chloro-3-fluorophenyl)-6-(4-chlorophenyl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole-2-carboxylic acid

The compound obtained in Step 12 above was reacted in the same way as inStep 13 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.91 (3H, d, J=7.3 Hz), 0.98 (3H, d, J=7.1Hz), 1.18 (1H, td, J=7.1, 0.9 Hz), 1.89 (3H, s), 5.98 (1H, s), 7.20-7.38(7H, m).

Reference Example 28

Step 1: tert-butyl[2-(4-chlorophenyl)-1-(6-chloropyridin-3-yl)-2-hydroxy-1-methylethyl]carbamate

4-chlorophenyl magnesium bromide instead of 4-chloro-3-fluorophenylmagnesium bromide was reacted in the same way as in Step 4 of ReferenceExample 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.45 (9H, brs), 1.57 (3H, s), 4.89 (1H, br),5.01 (1H, br), 5.17 (1H, br), 6.74-6.81 (1H, m), 7.09 (1H, brs),7.17-7.21 (1H, m), 7.28-7.36 (2H, m), 7.64 (1H, br), 8.40 (1H, br).

Step 2: tert-butyl[2-(4-chlorophenyl)-1-(6-chloropyridin-3-yl)-1-methyl-2-oxoethyl]carbamate

The compound obtained in Step 1 above was reacted in the same way as inStep 5 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.31 (9H, brs), 2.04 (3H, s), 6.45 (1H, br),7.28-7.35 (3H, m), 7.54 (2H, d, J=7.6 Hz), 7.71 (1H, br), 8.46 (1H, s).

Step 3: 2-amino-1-(4-chlorophenyl)-2-(6-chloropyridin-3-yl)propan-1-one

The compound obtained in Step 2 above was reacted in the same way as inStep 6 of Example 1 to give the title compound as a colorless oil.

MS (ESI) m/z: 295 [(M+1)]⁺.

Step 4:2-chloro-5-[4-(4-chlorophenyl)-3-methyl-1,1-dioxido-2,3-dihydro-1,2,5-thiadiazol-3-yl]pyridine

The compound obtained in Step 3 above was reacted in the same way as inStep 7 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 2.07 (3H, s), 7.53-7.62 (3H, m), 7.72-7.80(3H, m), 8.53 (1H, d, J=2.2 Hz), 9.11 (1H, br).

MS (ESI) m/z: 356 [(M+H)]⁺.

Step 5:(3S*,4R*)-2-chloro-5-[(3S,4R)-4-(4-chlorophenyl)-3-methyl-1,1-dioxido-1,2,5-thiadiazolidin-3-yl]pyridine

The compound obtained in Step 4 above was reacted in the same way as inStep 8 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.77 (3H, s), 4.95 (1H, d, J=5.0 Hz), 7.09(2H, d, J=8.2 Hz), 7.25 (2H, d, J=8.2 Hz), 7.32 (1H, d, J=8.5 Hz),7.48-7.50 (1H, m), 7.95 (1H, d, J=5.0 Hz), 8.01-8.06 (2H, m).

MS (ESI) m/z: 358 [(M+1)]⁺.

Step 6:(1R*,2S*)-1-(4-chlorophenyl)-2-(6-chloropyridin-3-yl)propane-1,2-diamine

The compound obtained in Step 5 above was reacted in the same way as inStep 9 of Example 1 to give the title compound as a pale yellow oil.

¹H-NMR (400 MHz, CDCl₃) δ: 1.52 (3H, s), 1.58 (4H, brs), 4.08 (1H, s),6.96 (2H, d, J=8.3 Hz), 7.17-7.22 (3H, m), 7.57 (1H, dd, J=8.4, 2.6 Hz),8.33 (1H, d, J=2.4 Hz).

MS (ESI) m/z: 296 [(M+1)]⁺.

Step 7:(1R,2S)-1-(4-chlorophenyl)-2-(6-chloropyridin-3-yl)propane-1,2-diamine

The compound obtained in Step 6 above was optically resolved in the sameway as in Step 10 of Example 1 to give the title compound as a yellowoil.

¹H-NMR (400 MHz, CDCl₃) δ: 1.52 (3H, s), 1.58 (4H, brs), 4.08 (1H, s),6.96 (2H, d, J=8.3 Hz), 7.17-7.22 (3H, m), 7.57 (1H, dd, J=8.4, 2.6 Hz),8.33 (1H, d, J=2.4 Hz).

[α]_(D)=+69.4° (c=2.0, chloroform, 24° C.)

Step 8:(4S,5R)-5-(4-chlorophenyl)-4-(6-chloropyridin-3-yl)-4-methylimidazolidine-2-thione

The compound obtained in Step 7 above was reacted in the same way as inStep 11 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 1.93 (3H, s), 5.02 (1H, s), 6.36 (1H, brs),6.70 (1H, brs), 6.85 (2H, d, J=8.5 Hz), 7.12 (1H, d, J=8.3 Hz), 7.15(2H, d, J=8.3 Hz), 7.25-7.30 (1H, m), 7.97 (1H, d, J=2.7 Hz).

MS (ESI) m/z: 338 [(M+1)]⁺.

Step 9: ethyl(5R,6S)-5-(4-chlorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole-2-carboxylate

The compound obtained in Step 8 above was reacted in the same way as inStep 12 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, CDCl₃) δ: 0.88 (3H, d, J=7.1 Hz), 1.01 (3H, d, J=7.1Hz), 1.33 (3H, t, J=7.1 Hz), 1.83 (3H, s), 3.32-3.43 (1H, m), 4.26 (2H,q, J=7.2 Hz), 5.12 (1H, s), 6.68-6.81 (2H, brm), 7.00 (1H, d, J=8.3 Hz),7.09 (2H, d, J=8.8 Hz), 7.49 (1H, dd, J=8.3, 2.7 Hz), 8.20 (1H, d, J=2.7Hz).

MS (ESI) m/z: 476 [(M+1)]+.

Step 10:(5R,6S)-5-(4-chlorophenyl)-6-(6-chloropyridin-3-yl)-3-isopropyl-6-methyl-5,6-dihydroimidazo[2,1-b][1,3]thiazole-2-carboxylicacid

The compound obtained in Step 9 above was reacted in the same way as inStep 13 of Example 1 to give the title compound as a colorless solid.

¹H-NMR (400 MHz, DMSO-d₆) δ: 0.76 (3H, d, J=7.1 Hz), 0.96 (3H, d, J=7.1Hz), 1.72 (3H, s), 5.70 (1H, s), 6.46-6.62 (2H, m), 7.15-7.29 (3H, m),7.65 (1H, dd, J=8.2, 2.3 Hz), 8.25 (1H, s).

MS (ESI) m/z: 448 [(M+1)]⁺.

Test Example 1 Mdm2/p53 Binding Assay

A protein dilution containing 6.25 nM each of His-p53 (fusion protein ofa p53 partial protein having p53 amino acids at positions 1 to 132, witha histidine protein) and GST-Mdm2 (fusion protein of a Mdm2 partialprotein, having Mdm2 amino acids at positions 25 to 108 with leucineresidue 33 substituted by glutamic acid, with glutathione transferase)proteins was prepared using a protein buffer solution (20 mM HEPES pH7.4, 150 mM NaCl, 0.1% BSA). This protein dilution was added in anamount of 8 μL/well to a 384-well plate (384-well low volume NBC,Corning Inc., catalog No: 3676).

Next, a test compound was diluted with DMSO to produce protein buffersolution containing 10% dilution, and this buffer solution was added inan amount of 4 μL/well to the plate.

Subsequently, a solution containing an XL665-labeled anti-His antibody(HTRF monoclonal anti-6HIS antibody labeled with XL665 (catalog No:61HISXLB), Schering/Cisbio Bioassays) and a europium (Eu)-labeledanti-GST antibody (HTRF monoclonal anti-GST antibody labeled witheuropium cryptate, Schering/Cisbio Bioassays, catalog No: 61GSTKLB) atconcentrations of 2.5 μg/mL and 0.325 μg/mL, respectively, was preparedusing an antibody diluting buffer solution (20 mM HEPES pH 7.4, 150 mMNaCl, 0.1% BSA, 0.5 M KF). These dilutions were added in an amount of 8μL/well (total reaction solution volume: 20 μl/well). Then, the platewas left at 25° C. for 1 hour.

Time-resolved fluorescence at 620 and 665 nm was measured at anexcitation wavelength of 320 nm using a plate reader (ARVOsx,PerkinElmer Co., Ltd. or PHERAstar, BMG LABTECH). Ratio (R) wascalculated using the measured values (RFU 620 nm and RFU 665 nm)according to the following formula:

R=(RFU 665 nm−BI−C×RFU 620 nm)/RFU 620 nm

BI: measured value at 665 nm of reaction solution (only each buffersolution) nonsupplemented with each protein, the compound, and theantibodies

C(correction factor)=(A−BI)/D

A and D: each measured value at 665 nm and 620 nm of reaction solutionsupplemented with only Eu-labeled anti-GST antibody solution.

The R value calculated from the well supplemented with His-p53,GST-Mdm2, the test compound, and each antibody was defined as R(sample). The R value calculated from the well supplemented withHis-p53, GST-Mdm2, and each antibody but without the test compound wasdefined as R (control). The R value calculated from the wellsupplemented with GST-Mdm2, the test compound, and each antibody butwithout His-p53 was defined as R (background). T/C was calculated fromthe formula shown below. An IC₅₀ value for Mdm2/p53 binding wascalculated by sigmoid fitting. The results are shown in Table 1.

T/C=(R(sample)−R(background))/(R(control)−R(background))

The results are shown in Table 20.

TABLE 20 IC50 (μM) Compound of Example 1  0.0028 Compound of Example 2 0.0022 Compound of Example 3  0.0039 Compound of Example 4  0.0025Compound of Example 5  0.0053 Compound of Example 6  0.0024 Compound ofExample 7  0.0036 Compound of Example 8  0.0022 Compound of Example 9 0.0041 Compound of Example 10 0.014 Compound of Example 11 0.0032Compound of Example 12 0.0050 Compound of Example 13 0.0054 Compound ofExample 14 0.016 Compound of Example 15 0.0027 Compound of Example 160.0023 Compound of Example 17 0.0033 Compound of Example 18 0.0016Compound of Example 19 0.0016 Compound of Example 20 0.0014 Compound ofExample 21 0.0022 Compound of Example 22 0.0026 Compound of Example 230.0016 Compound of Example 24 0.0012 Compound of Example 25 0.0017Compound of Example 26 0.0027 Compound of Example 27 0.0019 Compound ofExample 28 0.0031 Compound of Example 29 0.0026 Compound of Example 300.0031 Compound of Example 31 0.0031

Test Example 2 Cell Growth Inhibition Assay

A cell growth inhibition assay was conducted using human lungcancer-derived cell line NCI-H460 having wild-type p53.

NCI-H460 cells were suspended in a medium (RPMI1640 medium containing10% fetal bovine serum) and the suspension was inoculated in an amountof 500 cells/150 μL/well to a 96-well multiwell plate. A test compoundwas dissolved in DMSO and this solution was diluted with medium toprepare a sample solution (DMSO concentration: 1% or lower). On the nextday of inoculation, medium nonsupplemented with the test compound or thesample solution was added in an amount of 50 μL/well. The MTT assay wasconducted immediately after the medium was added in an amount of 50 μLon the next day of cell inoculation, and after the sample solution orthe medium was added to cells followed by culturing at 37° C. for 3 daysin a 5% CO₂ atmosphere. The MTT assay was conducted as shown below.

A 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, Sigma-Aldrich Co., M-2128) solution was prepared using aphosphate buffer solution (Dulbecco's Phosphate-buffered Saline). ThisMTT solution was added in an amount of 20 μL/well. Then, the plate wascultured at 37° C. for 4 hours in a 5% CO₂ atmosphere. The plate wascentrifuged at 1200 rpm for 5 minutes and then the culture supernatantwas removed by aspiration using a dispenser. DMSO was added in an amountof 150 μL/well to dissolve generated formazan. The plate was stirredusing a plate mixer for uniform color development from each well. Theabsorbance of each well was measured under conditions of OD 540 nm andreference 660 nm using a plate reader (SpectraMax PLUS384, MolecularDevices, Calif., USA).

The OD value measured on the day of adding the sample solution wasdefined as S. The OD value measured three days after addition of thesample solution was defined as T. The OD value measured three days afteraddition of the DMSO dilution was defined as C. T/C (%) was determinedat each concentration according to the calculation formula shown belowto prepare a dose response curve, from which 50% growth inhibitionconcentration (GI₅₀ value) was calculated.

T/C(%)=(T−S)/(C−S)×100

The compounds of Examples 1 to 6, 8 to 10, 15, 16, 18 to 25, 27, 29, and31 exhibited anti-cellular effect of GI₅₀ (μM)<0.4. The compounds ofExamples 7, 11 to 14, 17, 26, 28, and 30 exhibited anti-cellular effectof 0.4≦GI₅₀<2.5 (μM).

Test Example 3 Anti-Tumor Activity Test

A human osteosarcoma cell line SJSA-1 or SJSA-1-RE (cells in which a p53reporter gene was incorporated in SJSA-1) was subcutaneouslytransplanted to nude mice (BALB/C-nu/nu SLC, male, Japan SLC, Inc.). Atthe point in time when the tumor size reached approximately 100 to 200mm³, the mice were divided into groups (6 mice/group). A test compoundwas suspended in 0.5% methylcellulose solution and orally administeredtwice a day (bid) at a dose of 50 mg/kg for 4 consecutive days. After2-day drug holiday, the mice were dissected, the tumors were excised andthen their weights were measured.

The anti-tumor effect (IR (%)) was calculated according to the followingformula:

IR(%)=[1−(average tumor weight of compound-administered group/averagetumor weight of untreated control group)]×100.

The compound of Example 2 exhibited anti-tumor effect of 50<IR (%)<70.The compounds of Examples 3, 4, 6, and 9 exhibited anti-tumor effect of70<IR (%)<100.

Test Example 4 Metabolic Stability Test

100 μL of 100 mM phosphate buffer solution (pH 7.4) containing 3 μM testcompound was added to 100 μL of reaction solution containing 100 mMphosphate buffer solution (pH 7.4), 30 mM glucose 6-phosphate, 10 mMMgCl₂.6H₂O, 3 units/mL glucose 6-phosphate 1-dehydrogenase, and 0.3 to1.5 mgP/mL human liver microsomes and the mixture was incubated at 37°C. for 20 minutes. Then, 70 μL of 100 mM phosphate buffer solution (pH7.4) containing 3 mM NADP+ was added and the mixture was furtherincubated at 37° C. for 30 minutes to conduct a microsomal metabolismtest. The compound was quantified by the internal standard method usinga quadrupole mass spectrometer connected to a high performance liquidchromatography apparatus. The metabolic stability (residual percentageof compound: MS %) was determined according to the following formula:

MS(human)(%)=(peak area ratio of test compound after addition of NADP+and incubation for 30 minutes)/(peak area ratio of test compound beforeaddition of NADP+)×100.

(peak area ratio: peak area of test compound divided by that of internalstandard substance)

The results are shown in Table 21.

TABLE 21 MS (human) % Compound of Example 1  77 Compound of Example 2 100 Compound of Example 3  68 Compound of Example 4  100 Compound ofExample 5  ND Compound of Example 6  69 Compound of Example 7  100Compound of Example 8  95 Compound of Example 9  80 Compound of Example10 48 Compound of Example 11 100 Compound of Example 12 69 Compound ofExample 13 100 Compound of Example 14 100 Compound of Example 15 44Compound of Example 16 56 Compound of Example 17 44 Compound of Example18 6 Compound of Example 19 93 Compound of Example 20 36 Compound ofExample 21 96 Compound of Example 22 60 Compound of Example 23 24Compound of Example 24 89 Compound of Example 25 47 Compound of Example26 57 Compound of Example 27 91 Compound of Example 28 51 Compound ofExample 29 96 Compound of Example 30 62 Compound of Example 31 6 (ND:Not Determined)

1-19. (canceled)
 20. A method of treating cancer, comprisingadministering a compound according to formula (1):

or a pharmaceutically acceptable salt thereof, to a patient, wherein Ar₁represents a phenyl group optionally substituted with one or moresubstituents each independently selected from a halogen atom, a cyanogroup, and a C₁-C₆ alkyl group; Ar₂ represents a phenyl group optionallysubstituted with one or more substituents each independently selectedfrom a halogen atom, a C₁-C₆ alkyl group, and a cyano group; or apyridyl group optionally substituted with one or more substituents eachindependently selected from a halogen atom, a C₁-C₆ alkyl group, and acyano group; R¹ represents a C₁-C₆ alkyl group optionally substitutedwith one or more substituents each independently selected from a halogenatom, a hydroxy group, a C₁-C₆ alkoxy group, a carbamoyl group, an aminogroup, a C₁-C₆ alkanoyl group, and a cyano group; or a C₁-C₆ alkanoylgroup optionally substituted with one or more substituents eachindependently selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, and a cyano group; ahydrogen atom; or a hydroxy group; R² and R³ each independentlyrepresent a C₁-C₆ alkyl group optionally substituted with one or moresubstituents each independently selected from a halogen atom, a hydroxygroup, a C₁-C₆ alkoxy group, a carbamoyl group, an amino group, a C₁-C₆alkanoyl group, and a cyano group; a carboxy group; or a hydrogen atom;or R² and R³ may together form an oxo group; or R² and R³ together withthe carbon atoms to which R² and R³ are respectively bonded may form a3- to 5-membered saturated hydrocarbon ring in a spiro form; R⁴represents a C₁-C₆ alkyl group optionally substituted with one or moresubstituents each independently selected from a halogen atom, a hydroxygroup, a C₁-C₆ alkoxy group, a carbamoyl group, an amino group, a C₁-C₆alkanoyl group, and a cyano group; R⁵ represents a C₁-C₆ alkyl groupoptionally substituted with one or more substituents each independentlyselected from a halogen atom, a hydroxy group, a C₁-C₆ alkoxy group, acarbamoyl group, an amino group, a C₁-C₆ alkanoyl group, and a cyanogroup; R⁶ represents a halogen atom or a hydrogen atom; and R⁷represents a halogen atom.
 21. The method according to claim 20, whereinthe cancer is selected from lung cancer, breast cancer, prostate cancer,colon cancer, acute myeloid leukemia, malignant lymphoma,retinoblastoma, neuroblastoma, and sarcoma.
 22. The method according toclaim 20, wherein the cancer is selected from lung cancer, prostatecancer, colon cancer, acute myeloid leukemia, malignant lymphoma, andosteosarcoma.
 23. The method according to claim 20, wherein the compoundinhibits suppression of p53 transcription.
 24. The method according toclaim 20, wherein the compound prevents degradation of p53.
 25. Themethod according to claim 20, wherein the compound inhibits Mdm2-p53binding and ubiquitination of p53 by Mdm2.
 26. The method according toclaim 20, wherein the patient is a human.
 27. The method according toclaim 26, wherein the compound is administered in an amount of 0.01 to500 mg/kg body weight per day.
 28. The method according to claim 26,wherein the compound is administered in an amount of 0.1 to 100 mg/kgbody weight per day.
 29. The method according to claim 26, wherein thecompound is administered at least once per day.
 30. The method accordingto claim 26, wherein the compound is administered at least twice perday.
 31. The method according to claim 26, wherein the compound isadministered at least four times per day.
 32. The method according toclaim 20, wherein the compound is represented by general formula (2):

or a pharmaceutically acceptable salt thereof, and R¹, R², R³, R⁴, R⁵,R⁶, and R⁷ are as defined in claim
 20. 33. The method according to claim20, wherein the compound is selected from:

or a pharmaceutically acceptable salt thereof;

or a pharmaceutically acceptable salt thereof;

or a pharmaceutically acceptable salt thereof;

or a pharmaceutically acceptable salt thereof; and

or a pharmaceutically acceptable salt thereof.
 34. A method ofinhibiting cell growth, comprising administering a compound according toformula (1):

or a pharmaceutically acceptable salt thereof, to a patient, wherein Ar₁represents a phenyl group optionally substituted with one or moresubstituents each independently selected from a halogen atom, a cyanogroup, and a C₁-C₆ alkyl group; Ar₂ represents a phenyl group optionallysubstituted with one or more substituents each independently selectedfrom a halogen atom, a C₁-C₆ alkyl group, and a cyano group; or apyridyl group optionally substituted with one or more substituents eachindependently selected from a halogen atom, a C₁-C₆ alkyl group, and acyano group; R¹ represents a C₁-C₆ alkyl group optionally substitutedwith one or more substituents each independently selected from a halogenatom, a hydroxy group, a C₁-C₆ alkoxy group, a carbamoyl group, an aminogroup, a C₁-C₆ alkanoyl group, and a cyano group; or a C₁-C₆ alkanoylgroup optionally substituted with one or more substituents eachindependently selected from a halogen atom, a hydroxy group, a C₁-C₆alkoxy group, a carbamoyl group, an amino group, and a cyano group; ahydrogen atom; or a hydroxy group; R² and R³ each independentlyrepresent a C₁-C₆ alkyl group optionally substituted with one or moresubstituents each independently selected from a halogen atom, a hydroxygroup, a C₁-C₆ alkoxy group, a carbamoyl group, an amino group, a C₁-C₆alkanoyl group, and a cyano group; a carboxy group; or a hydrogen atom;or R² and R³ may together form an oxo group; or R² and R³ together withthe carbon atoms to which R² and R³ are respectively bonded may form a3- to 5-membered saturated hydrocarbon ring in a spiro form; R⁴represents a C₁-C₆ alkyl group optionally substituted with one or moresubstituents each independently selected from a halogen atom, a hydroxygroup, a C₁-C₆ alkoxy group, a carbamoyl group, an amino group, a C₁-C₆alkanoyl group, and a cyano group; R⁵ represents a C₁-C₆ alkyl groupoptionally substituted with one or more substituents each independentlyselected from a halogen atom, a hydroxy group, a C₁-C₆ alkoxy group, acarbamoyl group, an amino group, a C₁-C₆ alkanoyl group, and a cyanogroup; R⁶ represents a halogen atom or a hydrogen atom; and R⁷represents a halogen atom.
 35. The method according to claim 34, whereinthe compound inhibits suppression of p53 transcription.
 36. The methodaccording to claim 34, wherein the compound prevents degradation of p53.37. The method according to claim 34, wherein the compound inhibitsMdm2-p53 binding and ubiquitination of p53 by Mdm2.
 38. The methodaccording to claim 37, wherein the compound is administered in an amountselected from 0.01 to 500 mg/kg body weight per day and 0.1 to 100 mg/kgbody weight per day.
 39. The method according to claim 37, wherein thecompound is administered at least once per day, at least twice per day,or at least four times per day.